research article mir-136 modulates tgf- 1-induced...
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Research ArticlemiR-136 Modulates TGF-1205731-Induced Proliferation Arrest byTargeting PPP2R2A in Keratinocytes
Dianbao Zhang1 Jing Wang2 Zhe Wang3 Tao Zhang1 Ping Shi4 Xiliang Wang1
Feng Zhao1 Xiaoyu Liu1 Xuewen Lin1 and Xining Pang1
1Department of Stem Cells and Regenerative Medicine Key Laboratory of Cell Biology Ministry of Public Healthand Key Laboratory of Medical Cell Biology Ministry of Education China Medical University Shenyang 110001 China2Department of Anus amp Intestine Surgery The First Affiliated Hospital China Medical University Shenyang 110001 China3Department of Blood Transfusion Shengjing Hospital China Medical University Shenyang 110004 China4Department of General Practice The First Affiliated Hospital China Medical University Shenyang 110001 China
Correspondence should be addressed to Xining Pang pangxining126com
Received 14 June 2014 Accepted 23 September 2014
Academic Editor Paul J Higgins
Copyright copy 2015 Dianbao Zhang et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Keratinocytes proliferation is critical for the capacity to heal wounds and accumulating evidences have proved that dysregulation ofmicroRNAs is involved in proliferation of keratinocytes However the molecular mechanisms remain to be completely elucidatedHere we show that miR-136 was significantly decreased by TGF-1205731 treatment in HaCaT cells and normal human epidermalkeratinocytes (NHEK) and it was a Smad3-dependent manner By cell proliferation assay and cell cycle analysis we found thatreintroduction of miR-136 by transfection as well as PPP2R2A silencing counteracted TGF-120573-induced proliferation arrest inHaCaT cells Further PPP2R2A was verified as a direct target of miR-136 by dual-luciferase reporter assays and Western blottingThese data suggest that miR-136 may play an important role during TGF-1205731-induced proliferation arrest by targeting PPP2R2A inkeratinocytes which might represent a potential target for improving skin wound healing
1 Introduction
Keratinocytes proliferation is essential for the formation ofskin appendages and reepithelialization after wounding [1]and epidermal homeostasis depends on a balance of kerat-inocytes between proliferation in the basal layer and dif-ferentiationapoptosis in the superbasal layer [2ndash4] So farthese functions have been shown to be stimulated by manygrowth factors including transforming growth factor-1205731(TGF-1205731) [5 6] Moreover TGF-1205731 secreted by fibroblastsand keratinocytes would cause proliferation arrest of kerat-inocytes through activating the signaling pathways by bind-ing a receptor complex comprising two transmembrane ser-inethreonine kinases known as the type I and type II recep-tors [7] Thus exploring the regulation mechanisms of TGF-1205731-induced keratinocytes proliferation arrest is necessaryto understand skin development and wound repair and todesign novel therapeutic strategies for skin wound healing
MicroRNAs (miRNAs) represent a large family of con-served noncoding small RNAs (sim21 nucleotides long) whichhave emerged as key regulators of gene expression at both thetranscriptional and translational levels Recently increasingevidences suggest that miRNAs are responsible for regulatingproliferation and self-renewal of keratinocytes [8] Depletionof the miRNA processing enzyme Dicer in the epidermisresults in hyperproliferation [9] and several miRNAs havebeen shown to modulate keratinocytes proliferation such asmiR-34 family [10] miR-125b [11] miR-200 family [12] miR-203 [13] and miR-210 [14] Nonetheless the crucial role ofmiR-136 in the TGF-1205731-induced proliferation arrest is largelyunknown
PPP2R2A also known as B55120572 is a regulatory subunitof the protein phosphatase 2A (PP2A) which is one of thefour major serinethreonine (SerThr) phosphatases and isimplicated in the negative control of cell growth and divisioninmammalian cells [15 16]The crystal structure of PPP2R2A
Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 453518 8 pageshttpdxdoiorg1011552015453518
2 BioMed Research International
has provided insight on how the B subunit forms a pocketwith the catalytic subunit thatmay confer substrate specificity[17] PPP2R2A plays an important role in the modulationof the phosphorylation state of pocket proteins and mitoticCDK substrates throughout the cell cycle and in quiescentcells [18]
In the present study we identified and characterizedPPP2R2A as a functional downstream target of miR-136 andexamined the roles of miR-136 and PPP2R2A in TGF-1205731-induced proliferation arrest in keratinocytes
2 Materials and Methods
21 Cell Isolation and Culture Normal human epidermalkeratinocytes (NHEK) were isolated from foreskins (18ndash26years old 119899 = 3) usingDispase II (Roche IN USA) to removethe dermis from the epidermis followed by trypsinizationand culture in EpiLife serum-free medium with 60 120583M cal-cium supplemented with HKGS (Gibco NY USA) Writteninformed consentwas obtained fromall patients and approvalof the China Medical University Ethics Committee wasobtained
The immortalized human keratinocyte cell line HaCaTwas kindly provided by Chundi He Cos-7 cells were pur-chased from ATCC HaCaT cells and Cos-7 cells were cul-tured in DMEM (Hyclone MA USA) supplemented with10 fetal bovine serum All the cells were maintained understandard conditions at 37∘C and 5 CO
2in a humid atmo-
sphere For TGF-1205731 treatment NHEK or HaCaT cells wereplated at 3000 cellscm2 andmaintained for 24 h after whichtheywere serum-starved for another 24 hThen the cells werecultured in the absence or presence of TGF-1205731 (RampDSystemsMN USA) for 48 h and 72 h
22 Quantitative Real-Time PCR (qRT-PCR) Total RNA wasextracted using TRIzol reagents (Invitrogen CA USA)according to themanufacturerrsquos protocol To analyzemiR-136expression reverse transcription PCR was performed usingspecific stem-loop RT primers from a Hairpin-it miRNAsqPCRQuantitation Kit (GenePharma Shanghai China) andquantitative real-time PCR was performed using the same kiton Applied Biosystems 7500 system U6 was used as an inter-nal control To quantifymRNA level of PPP2R2A and Smad3reverse transcription PCR was applied using PrimeScriptRT Reagent Kit with gDNA Eraser (Takara Dalian China)and quantitative real-time PCR was performed using SYBRPremix Ex Taq (Takara Dalian China) GAPDHwas used asan internal control Fold changes of both miRNA andmRNAwere calculated using the 2minusΔΔCt method Each experimentwas performed in triplicateTheprimer sequenceswere as fol-lows 51015840-AACCCACTTCCTGCTTAGTTGAG-31015840 (forward)and 51015840-GAACACCACAGTGATGAATCCAC-31015840 (reverse)for human PPP2R2A 51015840-ACTACATCGGAGGGGAGGTC-31015840 (forward) and 51015840-TGCATCCTGGTGGGATCTTG-31015840(reverse) for human Smad3 51015840-GCACCGTCAAGGCTG-AGAAC-31015840 (forward) and 51015840-TGGTGAAGACGCCAG-TGGA-31015840 (reverse) for human GAPDH
23 miRNA and siRNA Transfection Cells cultured in 6-well plates were transfected at 30ndash50 confluence usingLipofectamine RNAiMAX Transfection Reagent (InvitrogenCA USA) with Opti-MEM I (Gibco NY USA) accordingto the manufacturerrsquos instructions Briefly 25 pmol of miR-136 mimics PPP2R2A siRNA (Santa Cruz CA USA) Smad3siRNA or negative control (NC) was used with 75 120583L of thetransfection reagent and the mediumwas replaced with freshmedium 24 h after transfection Smad3 siRNA miR-136mimics and NC were chemical synthesized by GenePharma(Shanghai China) and the sequences were as follows 51015840-GAGCCTGGTCAAGAAACTCAATT-31015840 for Smad3 siRNA51015840-ACUCCAUUUGUUUUGAUGAUGGA-31015840 for miR-136mimics and 51015840-UUCUCCGAACGUGUCACGUTT-31015840 forNC
24 Cell Proliferation Assay Cell proliferation was measuredusing Cell Counting Kit-8 (CCK-8 Dojindo KumamotoJapan) 10 120583L CCK-8 was added to cells maintained in 96-well plates and the cells were subsequently incubated for 2 h at37∘CThe absorbance of each sample at 450 nmwasmeasuredwith a reference wavelength of 630 nm All experiments wereperformed in triplicate and repeated three times
25 Cell CycleAnalysis Cell Cycle andApoptosisAnalysisKit(Beyotime Nantong China) was used for cell cycle analysisCells were fixed in 70 ethanol in PBS at minus20∘C for 12 hafter washing three times with cold PBS then the cells werewashed again and stained with 05mL of propidium iodide(PI) staining buffer (it contains 200mgmL RNase A and50 120583gmL PI) at 37∘C for 30min in the dark Analyses wereperformed on BD LSR flow cytometerThe experiments wererepeated three times
26 Dual-Luciferase Reporter Assays Bioinformatic analysisof microRNA target sites was performed using TargetScanHuman Release 62 (httpwwwtargetscanorg) Oligonu-cleotide pairs were designed to contain wild-type or mutantputative miRNA target sequences in 31015840-UTR of PPP2R2Awith proper overhangs and an internal KpnI site for cloneconfirmation Annealing and ligating these pairs into thepmirGLO Dual-Luciferase miRNA Target Expression Vectormaintained the miRNA target region in the correct 51015840 to31015840 orientation Oligonucleotide pairs were annealed (95∘Cfor 3min cooled to 37∘C over 60min and kept at 4∘C for30min) ligated with the SacI-XbaI digested and gel-purifiedpmirGLO vector and transformed into E coli DH5120572 Thepositive cloneswere identified by digestingminiprep-purifiedDNA with KpnI and verified by sequencing Cos-7 cellsplated on 24-well plates were cotransfected with 50 nmol ofeither miR-136mimics or NC oligos and with 200 ng reporterplasmid containing either wild-type or mutant target siteusing Lipofectamine 2000 Transfection Reagent accordingto the manufacturerrsquos instructions Cells were lysed and therelative luciferase activities were quantified at 48 h after trans-fection using Dual Glo Luciferase Assay System (PromegaWI USA) All transfections were carried out in triplicate
BioMed Research International 3
Table 1 Oligonucleotides for dual-luciferase reporter assays All oligonucleotides for 3 target sites were designed with proper overhangs forSacI and XbaI digestion Mutation sites were shown in italics and KpnI restriction enzyme sites were shown in bold
Name Oligonucleotides sequencewt site 1 sense 51015840-CGGTACCCAACACATTGTTATAGCTACATGGAGAAAGCTT-31015840
wt site 1 antisense 51015840-CTAGAAGCTTTCTCCATGTAGCTATAACAATGTGTTGGGTACCGAGCT-31015840
mut site 1 sense 51015840-CGGTACCCAACACATTGTTATAGCTACGGAACTAAAGCTT-31015840
mut site 1 antisense 51015840-CTAGAAGCTTTAGTTCCGTAGCTATAACAATGTGTTGGGTACCGAGCT-31015840
wt site 2 sense 51015840-CGGTACCACTGAATAAAAGTGACAAAGAATGGAGAATCTGT-31015840
wt site 2 antisense 51015840-CTAGACAGATTCTCCATTCTTTGTCACTTTTATTCAGTGGTACCGAGCT-31015840
mut site 2 sense 51015840-CGGTACCACTGAATAAAAGTGACAAAGCGGAACTAATCTGT-31015840
mut site 2 antisense 51015840-CTAGACAGATTAGTTCCGCTTTGTCACTTTTATTCAGTGGTACCGAGCT-31015840
wt site 3 sense 51015840-CGGTACCGTCAGTCTTGCAGAAACTTTAATGGAGAAGAAAT-31015840
wt site 3 antisense 51015840-CTAGATTTCTTCTCCATTAAAGTTTCTGCAAGACTGACGGTACCGAGCT-31015840
mut site 3 sense 51015840-CGGTACCGTCAGTCTTGCAGAAACTTTCGGAACTAAGAAAT-31015840
mut site 3 antisense 51015840-CTAGATTTCTTAGTTCCGAAAGTTTCTGCAAGACTGACGGTACCGAGCT-31015840
Theoligonucleotide sequences used for each reporter plasmidconstruction are listed in Table 1
27 Western Blot Protein was harvested using RIPA lysisbuffer (Dingguo Beijing China) supplemented with01mmol PMSF diluted with 5 times RSB and denatured at 95∘Cfor 10min Protein lysates were separated by 10 SDS-PAGEand transferred to PVDF membranes the membraneswere blocked in 5 nonfat milk for 1 h and then incubatedwith mouse anti-PPP2R2A (1 1000 CST MA USA) andmouse anti-GAPDH (1 5000 KangChen Shanghai China)antibodies overnight at 4∘C After incubation with HRP-con-jugated secondary antibody (1 5000 KangChen ShanghaiChina) protein bands were visualized using Amersham ECLPrime Western Blotting Detection Reagent (GE HealthcareNJ USA) on Fujifilm LAS-3000 Imager and quantified byImage J
28 Statistical Analysis Data were expressed as the mean plusmnSD from at least three independent experiments Studentrsquos t-test and one-way ANOVA were used to compare the differ-ences between groups All statistical analyses were performedusing SPSS 160 software (SPSS Inc USA) 119875 lt 005 wasconsidered statistically significant
3 Results
31 miR-136 Was Downregulated by TGF-1205731 in HaCaT andNHEKCells To investigate miR-136 expression changes dur-ing proliferation regulation of TGF-1205731 we performed quan-titative real-time PCR assay to measure miR-136 in ker-atinocytes treated with different concentration of TGF-1205731The result showed that the expression level of miR-136 inHaCaT decreased to 019-fold at 48 h after TGF-1205731 treatmentat 2 ngmL and 013-fold for 5 ngmL TGF-1205731 (Figure 1(a))To further confirm this result we isolated and culturedprimary epidermal keratinocytes from normal human skin
The results showed that miR-136 was also significantly down-regulated by TGF-1205731 treatment in NHEK (Figure 1(b))
32 miR-136 Suppression by TGF-1205731 Was Smad3-DependentIt was reported that Smad23 signaling as well as downstreamgene targets such as PAI-1 expression is required for TGF-120573 mediated cytostasis in HaCaT cells [19 20] To investigatewhether canonical Smad23 signaling is required for miR-136 repression during TGF-1205731-induced proliferation arrestin keratinocytes HaCaT cells were transfected with Smad3siRNA for 24 h and then treated with 2 ngmL TGF-1205731 for48 h By quantitative real-time PCR the mRNA level ofSmad3 was decreased to 02-fold at 48 h after transfection(Figure 1(c)) and in Smad3-silenced HaCaT cells TGF-1205731 treatment showed little effect on miR-136 expression(Figure 1(d)) These results indicated that miR-136 was sup-pressed by TGF-1205731 in a Smad3-dependent manner in HaCaTcells
33 Overexpression of miR-136 Overcomes TGF-1205731-InducedProliferation Arrest Previous studies showed that TGF-1205731inhibited cell proliferation in keratinocytes [21] to investigatewhether forced expression of miR-136 is able to modulateTGF-1205731-induced proliferation arrest of keratinocytes HaCaTcells were transfected with miR-136 mimics or miR-NCand subsequently stimulated with 2 ngmL TGF-1205731 for 72 hSuccessful increased expression of miR-136 was confirmedby qRT-PCR (Figure 2(a)) The results of cell proliferationassay showed that TGF-1205731 treatment inhibited HaCaT cellsproliferation effectively and led to gt20 proliferation sup-pression (Figure 2(b)) Moreover transfection of miR-136mimics could negatively regulate the proliferation inhibitionof TGF-1205731 and caused 29 proliferation enhancement Asshown in Figure 2(c) the cell subpopulation in S phasewas obviously decreased in TGF-1205731 treated cells comparedwith the control group while the cell number in S phasewas significantly increased in cells transfected with miR-136mimics versus miR-NC group Taken together these findings
4 BioMed Research International
Control0
02
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HaCaT
2ngmLTGF-1205731
5ngmLTGF-1205731
lowast
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expr
essio
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(a)
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02
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5ngmLTGF-1205731
Rela
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lowast
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iR-136
expr
essio
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(d)
Figure 1 miR-136 was downregulated by TGF-1205731 in a Smad3-dependent manner in HaCaT and NHEK cells The relative expression levelsof miR-136 were determined by quantitative real-time PCR (a) The expression of miR-136 was downregulated in HaCaT cells treated with2 ngmLor 5 ngmLTGF-1205731 for 48 h (b)Downregulation ofmiR-136was confirmed inNHEK treatedwithTGF-1205731 at the same concentrationsand time point (c) Smad3 silencing by siRNA was verified by quantitative real-time PCR (d) TGF-1205731-induced miR-136 repression wascounteracted by Smad3 knockdown Data were presented as mean plusmn SD values from three independent experiments Bars indicate SDlowast
119875 lt 005
indicated that miR-136 might act as a modulator of TGF-1205731-induced proliferation arrest in keratinocytes
34 PPP2R2A Was a Direct Target of miR-136 To elucidatethe underlyingmechanisms bywhichmiR-136 exerts its func-tion we exploredmiR-136 targets using the TargetScan bioin-formatics algorithmOur analysis revealed that PPP2R2Awasa potential target of miR-136 based on putative conservedtarget sequences at positions 149ndash155 712ndash719 and 1471ndash1478of the PPP2R2A 31015840-UTR (Figure 3(a)) To further examinewhether miR-136 directly targets PPP2R2A the luciferasereporters containing wild-type or mutant predicted miR-136 binding sites were cotransfected with miR-136 mimicsor NC into Cos-7 cells Luciferase assays were applied 48 hafter transfection and the results showed that compared
to NC transfection with miR-136 resulted in a significantdecrease in renillafirefly luciferase activity of wild-type site1 and site 2 reporter (Figures 3(b) and 3(c)) while therewas no significant decrease of wild-type site 3 reporter(Figure 3(d)) These results suggested that miR-136 repressedPPP2R2A through 2 specific 31015840-UTRbinding sites at positions149ndash155 and 712ndash719 Notably the expression of PPP2R2Ain HaCaT cells substantially decreased at 48 h after miR-136 transfection (Figure 3(e)) Taken together these resultsindicated that miR-136 negatively regulated PPP2R2A in aposttranscriptional manner in HaCaT cells
35 PPP2R2A Was Involved in TGF-1205731-Induced Prolifera-tion Arrest To further establish whether the counterac-tion of miR-136 overexpression against TGF-1205731-induced
BioMed Research International 5
0
5
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Control miR-136mimics
miR-NC
lowastRe
lativ
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-136
expr
essio
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(a)
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minus
minus
minus
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02
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val
ue (450
nm)
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S 3817
G0G1 4902G2M 1280 S 1858
G0G1 7424G2M 718
S 1359
G0G1 7958G2M 683 S 2219
G0G1 6676G2M 1105
TGF-1205731
(c)
Figure 2 Overexpression of miR-136 overcame TGF-1205731-induced proliferation arrest HaCaT cells were transfected with miR-136 mimicsor NC (a) The miR-136 level in HaCaT cells transfected with miR-136 mimics for 72 h was verified by qRT-PCR (b) Proliferation assaysin HaCaT cells stimulated with 2 ngmL TGF-1205731 for 24 h (c) Flow cytometry analysis revealed the cell cycle distribution Bars indicate SDlowast
119875 lt 005
proliferation arrest is mediated by repression of PPP2R2Athe expression of PPP2R2A in HaCaT cells treated with2 ngmL TGF-1205731 for 48 h was analyzed by Western blotand the proliferation of PPP2R2A knockdown HaCaT cellsstimulated with TGF-1205731 was assessed by proliferation assayand flow cytometry As shown in Figures 4(a) and 4(b)PPP2R2A in HaCaT cells was upregulated to about 2-foldby 2 ngmL TGF-1205731 treatment for 48 hours and in Figures4(c) and 4(d) knockdown of PPP2R2A clearly counteractedthe proliferation arrest induced by TGF-1205731 In addition thereduction of cell cycle progression at G1S transition inducedby TGF-1205731 was abrogated in PPP2R2A knockdown HaCaTcells The effects of PPP2R2A knockdown were similar tothose induced by miR-136 overexpression Taken togetherthese findings indicated that PPP2R2A was a functionallyimportant target of miR-136 and was involved in the TGF-1205731regulated proliferation of HaCaT cells
4 Discussion
Wound healing is a complex biological process during whichkeratinocyte proliferation and migration are crucial steps forthe rapid closure of the epidermis TGF-1205731 causes wound
margin contraction at the early stage of wound healing andit is responsible for scar formation [22] These processes arecontrolled by a network of biomolecules in a spatiotemporalmanner Several recent reports have indicated that miRNAsare involved in regulating keratinocyte proliferation duringwound healing [10ndash14] Here we aimed to clarify the biolog-ical role of miR-136 in keratinocytes proliferation regulationby TGF-1205731 The experiments showed significant reduction ofmiR-136 in keratinocytes treated with TGF-1205731 and canonicalSmad23 signaling pathway was involved Reintroduction ofmiR-136 by transient transfection as well as silencing bysiRNA of target PPP2R2A blocked TGF-1205731-induced prolif-eration arrest and increased the percentage of keratinocytesin the S phase of the cell cycle while reducing the percentageof those in the G0G1 phase Our results supported thenotion that TGF-1205731-induced proliferation arrest was partiallymediated by miR-136 reduction in HaCaT cells
There are several reports that miR-136 was implicated incell proliferation and played different roles in different typesof cells miR-136 is proposed to be a tumor suppressor inglioma and is capable of targeting the antiapoptosis genesAEG-1 and BCL-2 [23] However miR-136 was found totarget tumor suppressor PTEN in breast cancer cells [24]
6 BioMed Research International
hsa-miR-136
wt site 2mut site 2PtrMmuRnoOcu
hsa-miR-136
wt site 1mut site 1PtrMmuRnoOcu
ACAUUGUUAUAGCUACAUGGAGA
ACAUUGUUAUAGCUACGGAACUA
ACAUUGUUAUAGCUACAUGGAGA
CACAUUUGAUAGCCUCAUGGAGA
CACAUUUGAUAGCCACAUGGAGA
AUUUUUAUAUAGCUACAUGGAGA
1471ndash1478Site 3
712ndash719Site 2
149ndash155Site 1CDS
PPP2R2A
5998400
3998400
5998400
5998400
5998400
59984005998400
5998400
3998400
3998400
3998400
3998400
3998400
3998400
| ||| | |
AGGUAGUAGUUUUGUUUACCUCA
AAAAGUGACAAAG--AAUGGAGA
AAAAGUGACAAAG--CGGAACUA
AAAAGUGACAAAG--AAUGGAGA
AAAAGUGACAAAG--AAUGGAGA
AAAAGUGACAAAG--AAUGGAGA
AAAAGUGACAAAG--AAUGGAGA
5998400
998400 998400
5998400
5998400
5998400
59984005998400
5
3998400
3998400
3998400
3998400
3998400
3998400
| ||||| | | | | |
3 AGGUAGUAGUUUUGUUUACCUCA hsa-miR-136
wt site 3mut site 3PtrMmuRnoOcu
UCUUGCAGAAACUUUAAUGGAGA
UCUUGCAGAAACUUUCGGAACUA
UCUUGCAGAAACUUUAAUGGAGA
GUCCUGCGGAAGCUUUAUGGAGA
GUCCUGCGAAAGCUUUAUGGAGA
GUCUUGAAGAGCUUUAAUGGAGA
5998400
998400 998400
5998400
5998400
5998400
59984005998400
5
3998400399840039984003998400
39984003998400
||| || ||
3 AGGUAGUAGUUUUGUUUACCUCA
(a)
0020406081012
miR-NCmiR-136
+minus
+minus
minus
+minus
+wt site 1 mut site 1
lowast
Rela
tive l
ucife
rase
activ
ity
(b)
0020406081012
miR-NCmiR-136
+minus
+minus
minus
+minus
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lowastRe
lativ
e luc
ifera
se ac
tivity
(c)
0020406081012
miR-NCmiR-136
+minus
+minus
minus
+minus
+wt site 3 mut site 3
NS
Relat
ive l
ucife
rase
activ
ity
(d)
PPP2R2A
GAPDH
miR-136miR-NC mimics
(e)
Figure 3 PPP2R2A was a direct target of miR-136 (a) There were three potential miR-136 binding sites in PPP2R2A 31015840-UTR based on theTargetScan database the conservation of the miR-136 binding seed regions among different species was shown in shading and mutationswere shown in italics Fragments containing wild-type (wt) or mutant (mut) miR-136 binding sites in human PPP2R2A 31015840-UTR were cloneddownstreamof the luciferase reporter gene separately ((b)ndash(d)) Luciferase reporter assay (119899 = 3 for each group) Cos-7 cellswere cotransfectedwith a 31015840-UTR reporter construct and the miR-136 mimics or miR-NC and the results showed that site 1 and site 2 were the direct targets ofmiR-136 Luciferase activityrenilla activity was applied as the baseline control for the experiments using the same reporter Data representmean plusmn SD lowast119875 lt 005 (e) Western blot analyses of PPP2R2A expression in HaCaT cells transfected with miR-136 mimics or miR-NCGAPDH was used as loading control
PPP2R2A
GAPDH
Control TGF-1205731
(a)
0005101520253035 lowast
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ive P
PP2R
2A ex
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(b)
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Relat
ive P
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2A ex
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(c)
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02040608101214
TGF-1205731 +
lowast
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ue (450
nm)
PPP2R2A siRNA
(d)
Control
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0 40 80 120 160
S 3820
G0G1 4906G2M 1274
0 40 80 120 1600
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S 1367
G0G17889G2M 743
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PPP2R2A siRNA
S 2156
G0G1 6693G2M 1150
(e)
Figure 4 PPP2R2A was involved in TGF-1205731-induced proliferation arrest in HaCaT cells (a) Western blot analysis of PPP2R2A expression inresponse to TGF-1205731 treatment GAPDHwas used as internal control (b)Three independent results ofWestern blot were quantified by ImageJ (c) PPP2R2A silencing was verified by quantitative real-time PCR (d) Determination of cell viability with Cell Counting Kit-8 (e) Flowcytometry analysis of the cell cycle Bars indicate SD lowast119875 lt 005
BioMed Research International 7
TGF-1205731 PPP2R2AmiR-136
Keratinocytes proliferation
Figure 5 Schematic representation summarizing the roles of miR-136 and PPP2R2A during TGF-1205731-induced proliferation arrest Seetext for details
Recently results of others indicated that miR-136 enhancedphosphorylation of Erk12 through inhibition of PPP2R2Aexpression to promoted cell proliferation in human non-small cell lung cancer and the sequence at position 149ndash155 ofthe PPP2R2A 31015840-UTR was determined to be the target site ofmiR-136 [25] In this study we clarified miR-136 suppressedPPP2R2A expression by directly targeting two conservedregions at positions 149ndash155 and 712ndash719 in its 31015840-UTRFurthermore the protein level of PPP2R2A was significantlyreduced by miR-136 mimics transfection in HaCaT cellsTherefore we proposed that in the presence of TGF-1205731 miR-136 was a positive modulator of keratinocytes proliferationand PPP2R2A was a functional target of miR-136 via bindingto its 31015840-UTR region
In conclusion our results indicated that the miR-136treatment bypassed TGF-1205731-induced proliferation arrest atleast partially through downregulation of PPP2R2A expres-sion (Figure 5) Our data suggested that modulation ofkeratinocytes proliferation by miR-136 depends on the cel-lular environment especially in the presence of TGF-1205731which likely has major effects on the expression of miR-136target gene PPP2R2A Further studies are needed to deter-mine whether miR-136 based therapeutic strategies could beexploited to protect keratinocytes proliferation and functionin the condition of enhanced TGF-1205731 during wound healing
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Dianbao Zhang and Jing Wang contributed equally to thiswork
Acknowledgments
This work was funded by the National Basic ResearchProgram of China (2012CB518103) National Natural ScienceFoundation of China (81370883) Doctoral Fund of Ministryof Education of China (20132104110020) Science and Tech-nology Planning Project of Liaoning Province (2012225080)and Science and Technology Planning Project of Shenyang(F11-262-9-01) The authors thank Dr Hongchuan Xin andDr Ziwei Miao for critical reading and editing of this paper
References
[1] I Haase R Evans R Pofahl and F M Watt ldquoRegulation ofkeratinocyte shape migration and wound epithelialization byIGF-1 and EGF-dependent signalling pathwaysrdquo Journal of CellScience vol 116 no 15 pp 3227ndash3238 2003
[2] E Fuchs ldquoSkin stem cells rising to the surfacerdquo Journal of CellBiology vol 180 no 2 pp 273ndash284 2008
[3] C Pincelli andAMarconi ldquoKeratinocyte stem cells friends andfoesrdquo Journal of Cellular Physiology vol 225 no 2 pp 310ndash3152010
[4] J Nie X Fu and W Han ldquoMicroenvironment-dependenthomeostasis and differentiation of epidermal basal undiffer-entiated keratinocytes and their clinical applications in skinrepairrdquo Journal of the European Academy of Dermatology andVenereology vol 27 no 5 pp 531ndash535 2013
[5] S Guo and L A Dipietro ldquoFactors affecting wound healingrdquoJournal of Dental Research vol 89 no 3 pp 219ndash229 2010
[6] G S Schultz and AWysocki ldquoInteractions between extracellu-lar matrix and growth factors in wound healingrdquoWound Repairand Regeneration vol 17 no 2 pp 153ndash162 2009
[7] J S Kang C Liu andRDerynck ldquoNew regulatorymechanismsof TGF-120573 receptor functionrdquo Trends in Cell Biology vol 19 no8 pp 385ndash394 2009
[8] R Yi and E Fuchs ldquoMicroRNA-mediated control in the skinrdquoCell Death amp Differentiation vol 17 no 2 pp 229ndash235 2010
[9] M Teta Y S Choi T Okegbe et al ldquoInducible deletion of epi-dermal Dicer and Drosha reveals multiple functions for miR-NAs in postnatal skinrdquo Development vol 139 no 8 pp 1405ndash1416 2012
[10] D AntoniniM T Russo L de RosaMGorrese L del Vecchioand C Missero ldquoTranscriptional repression of miR-34 familycontributes to p63-mediated cell cycle progression in epidermalcellsrdquo Journal of Investigative Dermatology vol 130 no 5 pp1249ndash1257 2010
[11] N Xu P Brodin T Wei et al ldquoMiR-125b a MicroRNA down-regulated in psoriasis modulates keratinocyte proliferation bytargeting FGFR2rdquo Journal of Investigative Dermatology vol 131no 7 pp 1521ndash1529 2011
[12] Z Lin X Wang C Fewell J Cameron Q Yin and E KFlemington ldquoDifferential expression of the miR-200 familymicroRNAs in epithelial and B cells and regulation of Epstein-Barr virus reactivation by the miR-200 family member miR-429rdquo Journal of Virology vol 84 no 15 pp 7892ndash7897 2010
[13] A M Lena R Shalom-Feuerstein P R di Val Cervo et alldquomiR-203 represses ldquostemnessrdquo by repressingΔNp63rdquoCellDeathamp Differentiation vol 15 no 7 pp 1187ndash1195 2008
[14] S Biswas S Roy J Banerjee et al ldquoHypoxia inducible microR-NA 210 attenuates keratinocyte proliferation and impairs clo-sure in a murine model of ischemic woundsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 107 no 15 pp 6976ndash6981 2010
[15] M H Schmitz M Held V Janssens et al ldquoLive-cell imagingRNAi screen identifies PP2A-B55120572 and importin-120573 21 as keymitotic exit regulators in human cellsrdquo Nature Cell Biology vol12 no 9 pp 886ndash893 2010
[16] J Batut B Schmierer J Cao L A Raftery C S Hill and MHowell ldquoTwo highly related regulatory subunits of PP2A exertopposite effects on TGF-120573ActivinNodal signallingrdquo Develop-ment vol 135 no 17 pp 2927ndash2937 2008
[17] A Kurimchak and X Grana ldquoPP2A counterbalances phos-phorylation of pRB and mitotic proteins by multiple CDKs
8 BioMed Research International
potential implications for PP2A disruption in cancerrdquo Genes ampCancer vol 3 no 11-12 pp 739ndash748 2012
[18] G Jayadeva A Kurimchak J Garriga et al ldquoB55120572 PP2A hol-oenzymes modulate the phosphorylation status of the retino-blastomarelated protein p107 and its activationrdquo The Journal ofBiological Chemistry vol 285 no 39 pp 29863ndash29873 2010
[19] J M Overstreet R Samarakoon K K Meldrum and P JHiggins ldquoRedox control of p53 in the transcriptional regulationof TGF-1205731 target genes through SMAD cooperativityrdquo CellularSignalling vol 26 no 7 pp 1427ndash1436 2014
[20] M Motizuki K Isogaya K Miyake et al ldquoOligodendrocytetranscription factor 1 (Olig1) is a smad cofactor involved in cellmotility induced by transforming growth factor-120573rdquoThe Journalof Biological Chemistry vol 288 no 26 pp 18911ndash18922 2013
[21] K Rasanen and A Vaheri ldquoTGF-beta1 causes epithelial-mesenchymal transition in HaCaT derivatives but inducesexpression of COX-2 and migration only in benign not inmalignant keratinocytesrdquo Journal of Dermatological Science vol58 no 2 pp 97ndash104 2010
[22] G Viticchie A M Lena F Cianfarani et al ldquoMicroRNA-203contributes to skin re-epithelializationrdquo Cell Death amp Diseasevol 3 no 11 article e435 2012
[23] Y Yang J Wu H Guan et al ldquoMiR-136 promotes apoptosis ofglioma cells by targeting AEG-1 and Bcl-2rdquo FEBS Letters vol586 no 20 pp 3608ndash3612 2012
[24] D Y Lee Z Jeyapalan L Fang et al ldquoExpression of versican31015840-untranslated region modulates endogenous microrna func-tionsrdquo PLoS ONE vol 5 no 10 Article ID e13599 2010
[25] S Shen H Yue Y Li et al ldquoUpregulation of miR-136 in humannon-small cell lung cancer cells promotes Erk12 activation bytargeting PPP2R2Ardquo Tumor Biology vol 35 no 1 pp 631ndash6402014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
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Diabetes ResearchJournal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
2 BioMed Research International
has provided insight on how the B subunit forms a pocketwith the catalytic subunit thatmay confer substrate specificity[17] PPP2R2A plays an important role in the modulationof the phosphorylation state of pocket proteins and mitoticCDK substrates throughout the cell cycle and in quiescentcells [18]
In the present study we identified and characterizedPPP2R2A as a functional downstream target of miR-136 andexamined the roles of miR-136 and PPP2R2A in TGF-1205731-induced proliferation arrest in keratinocytes
2 Materials and Methods
21 Cell Isolation and Culture Normal human epidermalkeratinocytes (NHEK) were isolated from foreskins (18ndash26years old 119899 = 3) usingDispase II (Roche IN USA) to removethe dermis from the epidermis followed by trypsinizationand culture in EpiLife serum-free medium with 60 120583M cal-cium supplemented with HKGS (Gibco NY USA) Writteninformed consentwas obtained fromall patients and approvalof the China Medical University Ethics Committee wasobtained
The immortalized human keratinocyte cell line HaCaTwas kindly provided by Chundi He Cos-7 cells were pur-chased from ATCC HaCaT cells and Cos-7 cells were cul-tured in DMEM (Hyclone MA USA) supplemented with10 fetal bovine serum All the cells were maintained understandard conditions at 37∘C and 5 CO
2in a humid atmo-
sphere For TGF-1205731 treatment NHEK or HaCaT cells wereplated at 3000 cellscm2 andmaintained for 24 h after whichtheywere serum-starved for another 24 hThen the cells werecultured in the absence or presence of TGF-1205731 (RampDSystemsMN USA) for 48 h and 72 h
22 Quantitative Real-Time PCR (qRT-PCR) Total RNA wasextracted using TRIzol reagents (Invitrogen CA USA)according to themanufacturerrsquos protocol To analyzemiR-136expression reverse transcription PCR was performed usingspecific stem-loop RT primers from a Hairpin-it miRNAsqPCRQuantitation Kit (GenePharma Shanghai China) andquantitative real-time PCR was performed using the same kiton Applied Biosystems 7500 system U6 was used as an inter-nal control To quantifymRNA level of PPP2R2A and Smad3reverse transcription PCR was applied using PrimeScriptRT Reagent Kit with gDNA Eraser (Takara Dalian China)and quantitative real-time PCR was performed using SYBRPremix Ex Taq (Takara Dalian China) GAPDHwas used asan internal control Fold changes of both miRNA andmRNAwere calculated using the 2minusΔΔCt method Each experimentwas performed in triplicateTheprimer sequenceswere as fol-lows 51015840-AACCCACTTCCTGCTTAGTTGAG-31015840 (forward)and 51015840-GAACACCACAGTGATGAATCCAC-31015840 (reverse)for human PPP2R2A 51015840-ACTACATCGGAGGGGAGGTC-31015840 (forward) and 51015840-TGCATCCTGGTGGGATCTTG-31015840(reverse) for human Smad3 51015840-GCACCGTCAAGGCTG-AGAAC-31015840 (forward) and 51015840-TGGTGAAGACGCCAG-TGGA-31015840 (reverse) for human GAPDH
23 miRNA and siRNA Transfection Cells cultured in 6-well plates were transfected at 30ndash50 confluence usingLipofectamine RNAiMAX Transfection Reagent (InvitrogenCA USA) with Opti-MEM I (Gibco NY USA) accordingto the manufacturerrsquos instructions Briefly 25 pmol of miR-136 mimics PPP2R2A siRNA (Santa Cruz CA USA) Smad3siRNA or negative control (NC) was used with 75 120583L of thetransfection reagent and the mediumwas replaced with freshmedium 24 h after transfection Smad3 siRNA miR-136mimics and NC were chemical synthesized by GenePharma(Shanghai China) and the sequences were as follows 51015840-GAGCCTGGTCAAGAAACTCAATT-31015840 for Smad3 siRNA51015840-ACUCCAUUUGUUUUGAUGAUGGA-31015840 for miR-136mimics and 51015840-UUCUCCGAACGUGUCACGUTT-31015840 forNC
24 Cell Proliferation Assay Cell proliferation was measuredusing Cell Counting Kit-8 (CCK-8 Dojindo KumamotoJapan) 10 120583L CCK-8 was added to cells maintained in 96-well plates and the cells were subsequently incubated for 2 h at37∘CThe absorbance of each sample at 450 nmwasmeasuredwith a reference wavelength of 630 nm All experiments wereperformed in triplicate and repeated three times
25 Cell CycleAnalysis Cell Cycle andApoptosisAnalysisKit(Beyotime Nantong China) was used for cell cycle analysisCells were fixed in 70 ethanol in PBS at minus20∘C for 12 hafter washing three times with cold PBS then the cells werewashed again and stained with 05mL of propidium iodide(PI) staining buffer (it contains 200mgmL RNase A and50 120583gmL PI) at 37∘C for 30min in the dark Analyses wereperformed on BD LSR flow cytometerThe experiments wererepeated three times
26 Dual-Luciferase Reporter Assays Bioinformatic analysisof microRNA target sites was performed using TargetScanHuman Release 62 (httpwwwtargetscanorg) Oligonu-cleotide pairs were designed to contain wild-type or mutantputative miRNA target sequences in 31015840-UTR of PPP2R2Awith proper overhangs and an internal KpnI site for cloneconfirmation Annealing and ligating these pairs into thepmirGLO Dual-Luciferase miRNA Target Expression Vectormaintained the miRNA target region in the correct 51015840 to31015840 orientation Oligonucleotide pairs were annealed (95∘Cfor 3min cooled to 37∘C over 60min and kept at 4∘C for30min) ligated with the SacI-XbaI digested and gel-purifiedpmirGLO vector and transformed into E coli DH5120572 Thepositive cloneswere identified by digestingminiprep-purifiedDNA with KpnI and verified by sequencing Cos-7 cellsplated on 24-well plates were cotransfected with 50 nmol ofeither miR-136mimics or NC oligos and with 200 ng reporterplasmid containing either wild-type or mutant target siteusing Lipofectamine 2000 Transfection Reagent accordingto the manufacturerrsquos instructions Cells were lysed and therelative luciferase activities were quantified at 48 h after trans-fection using Dual Glo Luciferase Assay System (PromegaWI USA) All transfections were carried out in triplicate
BioMed Research International 3
Table 1 Oligonucleotides for dual-luciferase reporter assays All oligonucleotides for 3 target sites were designed with proper overhangs forSacI and XbaI digestion Mutation sites were shown in italics and KpnI restriction enzyme sites were shown in bold
Name Oligonucleotides sequencewt site 1 sense 51015840-CGGTACCCAACACATTGTTATAGCTACATGGAGAAAGCTT-31015840
wt site 1 antisense 51015840-CTAGAAGCTTTCTCCATGTAGCTATAACAATGTGTTGGGTACCGAGCT-31015840
mut site 1 sense 51015840-CGGTACCCAACACATTGTTATAGCTACGGAACTAAAGCTT-31015840
mut site 1 antisense 51015840-CTAGAAGCTTTAGTTCCGTAGCTATAACAATGTGTTGGGTACCGAGCT-31015840
wt site 2 sense 51015840-CGGTACCACTGAATAAAAGTGACAAAGAATGGAGAATCTGT-31015840
wt site 2 antisense 51015840-CTAGACAGATTCTCCATTCTTTGTCACTTTTATTCAGTGGTACCGAGCT-31015840
mut site 2 sense 51015840-CGGTACCACTGAATAAAAGTGACAAAGCGGAACTAATCTGT-31015840
mut site 2 antisense 51015840-CTAGACAGATTAGTTCCGCTTTGTCACTTTTATTCAGTGGTACCGAGCT-31015840
wt site 3 sense 51015840-CGGTACCGTCAGTCTTGCAGAAACTTTAATGGAGAAGAAAT-31015840
wt site 3 antisense 51015840-CTAGATTTCTTCTCCATTAAAGTTTCTGCAAGACTGACGGTACCGAGCT-31015840
mut site 3 sense 51015840-CGGTACCGTCAGTCTTGCAGAAACTTTCGGAACTAAGAAAT-31015840
mut site 3 antisense 51015840-CTAGATTTCTTAGTTCCGAAAGTTTCTGCAAGACTGACGGTACCGAGCT-31015840
Theoligonucleotide sequences used for each reporter plasmidconstruction are listed in Table 1
27 Western Blot Protein was harvested using RIPA lysisbuffer (Dingguo Beijing China) supplemented with01mmol PMSF diluted with 5 times RSB and denatured at 95∘Cfor 10min Protein lysates were separated by 10 SDS-PAGEand transferred to PVDF membranes the membraneswere blocked in 5 nonfat milk for 1 h and then incubatedwith mouse anti-PPP2R2A (1 1000 CST MA USA) andmouse anti-GAPDH (1 5000 KangChen Shanghai China)antibodies overnight at 4∘C After incubation with HRP-con-jugated secondary antibody (1 5000 KangChen ShanghaiChina) protein bands were visualized using Amersham ECLPrime Western Blotting Detection Reagent (GE HealthcareNJ USA) on Fujifilm LAS-3000 Imager and quantified byImage J
28 Statistical Analysis Data were expressed as the mean plusmnSD from at least three independent experiments Studentrsquos t-test and one-way ANOVA were used to compare the differ-ences between groups All statistical analyses were performedusing SPSS 160 software (SPSS Inc USA) 119875 lt 005 wasconsidered statistically significant
3 Results
31 miR-136 Was Downregulated by TGF-1205731 in HaCaT andNHEKCells To investigate miR-136 expression changes dur-ing proliferation regulation of TGF-1205731 we performed quan-titative real-time PCR assay to measure miR-136 in ker-atinocytes treated with different concentration of TGF-1205731The result showed that the expression level of miR-136 inHaCaT decreased to 019-fold at 48 h after TGF-1205731 treatmentat 2 ngmL and 013-fold for 5 ngmL TGF-1205731 (Figure 1(a))To further confirm this result we isolated and culturedprimary epidermal keratinocytes from normal human skin
The results showed that miR-136 was also significantly down-regulated by TGF-1205731 treatment in NHEK (Figure 1(b))
32 miR-136 Suppression by TGF-1205731 Was Smad3-DependentIt was reported that Smad23 signaling as well as downstreamgene targets such as PAI-1 expression is required for TGF-120573 mediated cytostasis in HaCaT cells [19 20] To investigatewhether canonical Smad23 signaling is required for miR-136 repression during TGF-1205731-induced proliferation arrestin keratinocytes HaCaT cells were transfected with Smad3siRNA for 24 h and then treated with 2 ngmL TGF-1205731 for48 h By quantitative real-time PCR the mRNA level ofSmad3 was decreased to 02-fold at 48 h after transfection(Figure 1(c)) and in Smad3-silenced HaCaT cells TGF-1205731 treatment showed little effect on miR-136 expression(Figure 1(d)) These results indicated that miR-136 was sup-pressed by TGF-1205731 in a Smad3-dependent manner in HaCaTcells
33 Overexpression of miR-136 Overcomes TGF-1205731-InducedProliferation Arrest Previous studies showed that TGF-1205731inhibited cell proliferation in keratinocytes [21] to investigatewhether forced expression of miR-136 is able to modulateTGF-1205731-induced proliferation arrest of keratinocytes HaCaTcells were transfected with miR-136 mimics or miR-NCand subsequently stimulated with 2 ngmL TGF-1205731 for 72 hSuccessful increased expression of miR-136 was confirmedby qRT-PCR (Figure 2(a)) The results of cell proliferationassay showed that TGF-1205731 treatment inhibited HaCaT cellsproliferation effectively and led to gt20 proliferation sup-pression (Figure 2(b)) Moreover transfection of miR-136mimics could negatively regulate the proliferation inhibitionof TGF-1205731 and caused 29 proliferation enhancement Asshown in Figure 2(c) the cell subpopulation in S phasewas obviously decreased in TGF-1205731 treated cells comparedwith the control group while the cell number in S phasewas significantly increased in cells transfected with miR-136mimics versus miR-NC group Taken together these findings
4 BioMed Research International
Control0
02
04
06
08
10
12
HaCaT
2ngmLTGF-1205731
5ngmLTGF-1205731
lowast
lowast
Rela
tive m
iR-136
expr
essio
n
(a)
NHEK
lowast
lowast
Control0
02
04
06
08
10
12
2ngmLTGF-1205731
5ngmLTGF-1205731
Rela
tive m
iR-136
expr
essio
n
(b)
Control0
02
04
06
08
10
12
NC Smad3 siRNA
lowast
Relat
ive S
mad
3 ex
pres
sion
(c)
Smad3 siRNA
Smad3 siRNA
00
02
04
06
08
10
12
TGF-1205731
lowast
lowast
Control+
TGF-1205731+
NC
Rela
tive m
iR-136
expr
essio
n
(d)
Figure 1 miR-136 was downregulated by TGF-1205731 in a Smad3-dependent manner in HaCaT and NHEK cells The relative expression levelsof miR-136 were determined by quantitative real-time PCR (a) The expression of miR-136 was downregulated in HaCaT cells treated with2 ngmLor 5 ngmLTGF-1205731 for 48 h (b)Downregulation ofmiR-136was confirmed inNHEK treatedwithTGF-1205731 at the same concentrationsand time point (c) Smad3 silencing by siRNA was verified by quantitative real-time PCR (d) TGF-1205731-induced miR-136 repression wascounteracted by Smad3 knockdown Data were presented as mean plusmn SD values from three independent experiments Bars indicate SDlowast
119875 lt 005
indicated that miR-136 might act as a modulator of TGF-1205731-induced proliferation arrest in keratinocytes
34 PPP2R2A Was a Direct Target of miR-136 To elucidatethe underlyingmechanisms bywhichmiR-136 exerts its func-tion we exploredmiR-136 targets using the TargetScan bioin-formatics algorithmOur analysis revealed that PPP2R2Awasa potential target of miR-136 based on putative conservedtarget sequences at positions 149ndash155 712ndash719 and 1471ndash1478of the PPP2R2A 31015840-UTR (Figure 3(a)) To further examinewhether miR-136 directly targets PPP2R2A the luciferasereporters containing wild-type or mutant predicted miR-136 binding sites were cotransfected with miR-136 mimicsor NC into Cos-7 cells Luciferase assays were applied 48 hafter transfection and the results showed that compared
to NC transfection with miR-136 resulted in a significantdecrease in renillafirefly luciferase activity of wild-type site1 and site 2 reporter (Figures 3(b) and 3(c)) while therewas no significant decrease of wild-type site 3 reporter(Figure 3(d)) These results suggested that miR-136 repressedPPP2R2A through 2 specific 31015840-UTRbinding sites at positions149ndash155 and 712ndash719 Notably the expression of PPP2R2Ain HaCaT cells substantially decreased at 48 h after miR-136 transfection (Figure 3(e)) Taken together these resultsindicated that miR-136 negatively regulated PPP2R2A in aposttranscriptional manner in HaCaT cells
35 PPP2R2A Was Involved in TGF-1205731-Induced Prolifera-tion Arrest To further establish whether the counterac-tion of miR-136 overexpression against TGF-1205731-induced
BioMed Research International 5
0
5
10
15
20
25
Control miR-136mimics
miR-NC
lowastRe
lativ
e miR
-136
expr
essio
n
(a)
TGF-1205731miR-NCmiR-136
minus
minus
minus
++minus
+minus
minus
+minus
+
0
02
04
06
08
10
12
14 lowast
lowast
OD
val
ue (450
nm)
(b)
0
200
400
600
100
300
500
0 40 80 120 160Control
0
200
400
600
100
300
500
0 40 80 120 1600
200
400
600
0 40 80 120 160miR-NC + TGF-1205731
0
200
400
600
100
300
500
0 40 80 120 160miR-136 + TGF-1205731
S 3817
G0G1 4902G2M 1280 S 1858
G0G1 7424G2M 718
S 1359
G0G1 7958G2M 683 S 2219
G0G1 6676G2M 1105
TGF-1205731
(c)
Figure 2 Overexpression of miR-136 overcame TGF-1205731-induced proliferation arrest HaCaT cells were transfected with miR-136 mimicsor NC (a) The miR-136 level in HaCaT cells transfected with miR-136 mimics for 72 h was verified by qRT-PCR (b) Proliferation assaysin HaCaT cells stimulated with 2 ngmL TGF-1205731 for 24 h (c) Flow cytometry analysis revealed the cell cycle distribution Bars indicate SDlowast
119875 lt 005
proliferation arrest is mediated by repression of PPP2R2Athe expression of PPP2R2A in HaCaT cells treated with2 ngmL TGF-1205731 for 48 h was analyzed by Western blotand the proliferation of PPP2R2A knockdown HaCaT cellsstimulated with TGF-1205731 was assessed by proliferation assayand flow cytometry As shown in Figures 4(a) and 4(b)PPP2R2A in HaCaT cells was upregulated to about 2-foldby 2 ngmL TGF-1205731 treatment for 48 hours and in Figures4(c) and 4(d) knockdown of PPP2R2A clearly counteractedthe proliferation arrest induced by TGF-1205731 In addition thereduction of cell cycle progression at G1S transition inducedby TGF-1205731 was abrogated in PPP2R2A knockdown HaCaTcells The effects of PPP2R2A knockdown were similar tothose induced by miR-136 overexpression Taken togetherthese findings indicated that PPP2R2A was a functionallyimportant target of miR-136 and was involved in the TGF-1205731regulated proliferation of HaCaT cells
4 Discussion
Wound healing is a complex biological process during whichkeratinocyte proliferation and migration are crucial steps forthe rapid closure of the epidermis TGF-1205731 causes wound
margin contraction at the early stage of wound healing andit is responsible for scar formation [22] These processes arecontrolled by a network of biomolecules in a spatiotemporalmanner Several recent reports have indicated that miRNAsare involved in regulating keratinocyte proliferation duringwound healing [10ndash14] Here we aimed to clarify the biolog-ical role of miR-136 in keratinocytes proliferation regulationby TGF-1205731 The experiments showed significant reduction ofmiR-136 in keratinocytes treated with TGF-1205731 and canonicalSmad23 signaling pathway was involved Reintroduction ofmiR-136 by transient transfection as well as silencing bysiRNA of target PPP2R2A blocked TGF-1205731-induced prolif-eration arrest and increased the percentage of keratinocytesin the S phase of the cell cycle while reducing the percentageof those in the G0G1 phase Our results supported thenotion that TGF-1205731-induced proliferation arrest was partiallymediated by miR-136 reduction in HaCaT cells
There are several reports that miR-136 was implicated incell proliferation and played different roles in different typesof cells miR-136 is proposed to be a tumor suppressor inglioma and is capable of targeting the antiapoptosis genesAEG-1 and BCL-2 [23] However miR-136 was found totarget tumor suppressor PTEN in breast cancer cells [24]
6 BioMed Research International
hsa-miR-136
wt site 2mut site 2PtrMmuRnoOcu
hsa-miR-136
wt site 1mut site 1PtrMmuRnoOcu
ACAUUGUUAUAGCUACAUGGAGA
ACAUUGUUAUAGCUACGGAACUA
ACAUUGUUAUAGCUACAUGGAGA
CACAUUUGAUAGCCUCAUGGAGA
CACAUUUGAUAGCCACAUGGAGA
AUUUUUAUAUAGCUACAUGGAGA
1471ndash1478Site 3
712ndash719Site 2
149ndash155Site 1CDS
PPP2R2A
5998400
3998400
5998400
5998400
5998400
59984005998400
5998400
3998400
3998400
3998400
3998400
3998400
3998400
| ||| | |
AGGUAGUAGUUUUGUUUACCUCA
AAAAGUGACAAAG--AAUGGAGA
AAAAGUGACAAAG--CGGAACUA
AAAAGUGACAAAG--AAUGGAGA
AAAAGUGACAAAG--AAUGGAGA
AAAAGUGACAAAG--AAUGGAGA
AAAAGUGACAAAG--AAUGGAGA
5998400
998400 998400
5998400
5998400
5998400
59984005998400
5
3998400
3998400
3998400
3998400
3998400
3998400
| ||||| | | | | |
3 AGGUAGUAGUUUUGUUUACCUCA hsa-miR-136
wt site 3mut site 3PtrMmuRnoOcu
UCUUGCAGAAACUUUAAUGGAGA
UCUUGCAGAAACUUUCGGAACUA
UCUUGCAGAAACUUUAAUGGAGA
GUCCUGCGGAAGCUUUAUGGAGA
GUCCUGCGAAAGCUUUAUGGAGA
GUCUUGAAGAGCUUUAAUGGAGA
5998400
998400 998400
5998400
5998400
5998400
59984005998400
5
3998400399840039984003998400
39984003998400
||| || ||
3 AGGUAGUAGUUUUGUUUACCUCA
(a)
0020406081012
miR-NCmiR-136
+minus
+minus
minus
+minus
+wt site 1 mut site 1
lowast
Rela
tive l
ucife
rase
activ
ity
(b)
0020406081012
miR-NCmiR-136
+minus
+minus
minus
+minus
+wt site 2 mut site 2
lowastRe
lativ
e luc
ifera
se ac
tivity
(c)
0020406081012
miR-NCmiR-136
+minus
+minus
minus
+minus
+wt site 3 mut site 3
NS
Relat
ive l
ucife
rase
activ
ity
(d)
PPP2R2A
GAPDH
miR-136miR-NC mimics
(e)
Figure 3 PPP2R2A was a direct target of miR-136 (a) There were three potential miR-136 binding sites in PPP2R2A 31015840-UTR based on theTargetScan database the conservation of the miR-136 binding seed regions among different species was shown in shading and mutationswere shown in italics Fragments containing wild-type (wt) or mutant (mut) miR-136 binding sites in human PPP2R2A 31015840-UTR were cloneddownstreamof the luciferase reporter gene separately ((b)ndash(d)) Luciferase reporter assay (119899 = 3 for each group) Cos-7 cellswere cotransfectedwith a 31015840-UTR reporter construct and the miR-136 mimics or miR-NC and the results showed that site 1 and site 2 were the direct targets ofmiR-136 Luciferase activityrenilla activity was applied as the baseline control for the experiments using the same reporter Data representmean plusmn SD lowast119875 lt 005 (e) Western blot analyses of PPP2R2A expression in HaCaT cells transfected with miR-136 mimics or miR-NCGAPDH was used as loading control
PPP2R2A
GAPDH
Control TGF-1205731
(a)
0005101520253035 lowast
Control TGF-1205731Relat
ive P
PP2R
2A ex
pres
sion
(b)
Control NC PPP2R2A00020406081012 lowast
siRNA
Relat
ive P
PP2R
2A ex
pres
sion
(c)
Control0
02040608101214
TGF-1205731 +
lowast
TGF-1205731+
NC
OD
val
ue (450
nm)
PPP2R2A siRNA
(d)
Control
0
200
400
600
100
300
500
0 40 80 120 160
S 3820
G0G1 4906G2M 1274
0 40 80 120 1600
200
400
600
TGF-1205731 + NC
S 1367
G0G17889G2M 743
0
200
400
600
0 40 80 120 160TGF-1205731 +
PPP2R2A siRNA
S 2156
G0G1 6693G2M 1150
(e)
Figure 4 PPP2R2A was involved in TGF-1205731-induced proliferation arrest in HaCaT cells (a) Western blot analysis of PPP2R2A expression inresponse to TGF-1205731 treatment GAPDHwas used as internal control (b)Three independent results ofWestern blot were quantified by ImageJ (c) PPP2R2A silencing was verified by quantitative real-time PCR (d) Determination of cell viability with Cell Counting Kit-8 (e) Flowcytometry analysis of the cell cycle Bars indicate SD lowast119875 lt 005
BioMed Research International 7
TGF-1205731 PPP2R2AmiR-136
Keratinocytes proliferation
Figure 5 Schematic representation summarizing the roles of miR-136 and PPP2R2A during TGF-1205731-induced proliferation arrest Seetext for details
Recently results of others indicated that miR-136 enhancedphosphorylation of Erk12 through inhibition of PPP2R2Aexpression to promoted cell proliferation in human non-small cell lung cancer and the sequence at position 149ndash155 ofthe PPP2R2A 31015840-UTR was determined to be the target site ofmiR-136 [25] In this study we clarified miR-136 suppressedPPP2R2A expression by directly targeting two conservedregions at positions 149ndash155 and 712ndash719 in its 31015840-UTRFurthermore the protein level of PPP2R2A was significantlyreduced by miR-136 mimics transfection in HaCaT cellsTherefore we proposed that in the presence of TGF-1205731 miR-136 was a positive modulator of keratinocytes proliferationand PPP2R2A was a functional target of miR-136 via bindingto its 31015840-UTR region
In conclusion our results indicated that the miR-136treatment bypassed TGF-1205731-induced proliferation arrest atleast partially through downregulation of PPP2R2A expres-sion (Figure 5) Our data suggested that modulation ofkeratinocytes proliferation by miR-136 depends on the cel-lular environment especially in the presence of TGF-1205731which likely has major effects on the expression of miR-136target gene PPP2R2A Further studies are needed to deter-mine whether miR-136 based therapeutic strategies could beexploited to protect keratinocytes proliferation and functionin the condition of enhanced TGF-1205731 during wound healing
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Dianbao Zhang and Jing Wang contributed equally to thiswork
Acknowledgments
This work was funded by the National Basic ResearchProgram of China (2012CB518103) National Natural ScienceFoundation of China (81370883) Doctoral Fund of Ministryof Education of China (20132104110020) Science and Tech-nology Planning Project of Liaoning Province (2012225080)and Science and Technology Planning Project of Shenyang(F11-262-9-01) The authors thank Dr Hongchuan Xin andDr Ziwei Miao for critical reading and editing of this paper
References
[1] I Haase R Evans R Pofahl and F M Watt ldquoRegulation ofkeratinocyte shape migration and wound epithelialization byIGF-1 and EGF-dependent signalling pathwaysrdquo Journal of CellScience vol 116 no 15 pp 3227ndash3238 2003
[2] E Fuchs ldquoSkin stem cells rising to the surfacerdquo Journal of CellBiology vol 180 no 2 pp 273ndash284 2008
[3] C Pincelli andAMarconi ldquoKeratinocyte stem cells friends andfoesrdquo Journal of Cellular Physiology vol 225 no 2 pp 310ndash3152010
[4] J Nie X Fu and W Han ldquoMicroenvironment-dependenthomeostasis and differentiation of epidermal basal undiffer-entiated keratinocytes and their clinical applications in skinrepairrdquo Journal of the European Academy of Dermatology andVenereology vol 27 no 5 pp 531ndash535 2013
[5] S Guo and L A Dipietro ldquoFactors affecting wound healingrdquoJournal of Dental Research vol 89 no 3 pp 219ndash229 2010
[6] G S Schultz and AWysocki ldquoInteractions between extracellu-lar matrix and growth factors in wound healingrdquoWound Repairand Regeneration vol 17 no 2 pp 153ndash162 2009
[7] J S Kang C Liu andRDerynck ldquoNew regulatorymechanismsof TGF-120573 receptor functionrdquo Trends in Cell Biology vol 19 no8 pp 385ndash394 2009
[8] R Yi and E Fuchs ldquoMicroRNA-mediated control in the skinrdquoCell Death amp Differentiation vol 17 no 2 pp 229ndash235 2010
[9] M Teta Y S Choi T Okegbe et al ldquoInducible deletion of epi-dermal Dicer and Drosha reveals multiple functions for miR-NAs in postnatal skinrdquo Development vol 139 no 8 pp 1405ndash1416 2012
[10] D AntoniniM T Russo L de RosaMGorrese L del Vecchioand C Missero ldquoTranscriptional repression of miR-34 familycontributes to p63-mediated cell cycle progression in epidermalcellsrdquo Journal of Investigative Dermatology vol 130 no 5 pp1249ndash1257 2010
[11] N Xu P Brodin T Wei et al ldquoMiR-125b a MicroRNA down-regulated in psoriasis modulates keratinocyte proliferation bytargeting FGFR2rdquo Journal of Investigative Dermatology vol 131no 7 pp 1521ndash1529 2011
[12] Z Lin X Wang C Fewell J Cameron Q Yin and E KFlemington ldquoDifferential expression of the miR-200 familymicroRNAs in epithelial and B cells and regulation of Epstein-Barr virus reactivation by the miR-200 family member miR-429rdquo Journal of Virology vol 84 no 15 pp 7892ndash7897 2010
[13] A M Lena R Shalom-Feuerstein P R di Val Cervo et alldquomiR-203 represses ldquostemnessrdquo by repressingΔNp63rdquoCellDeathamp Differentiation vol 15 no 7 pp 1187ndash1195 2008
[14] S Biswas S Roy J Banerjee et al ldquoHypoxia inducible microR-NA 210 attenuates keratinocyte proliferation and impairs clo-sure in a murine model of ischemic woundsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 107 no 15 pp 6976ndash6981 2010
[15] M H Schmitz M Held V Janssens et al ldquoLive-cell imagingRNAi screen identifies PP2A-B55120572 and importin-120573 21 as keymitotic exit regulators in human cellsrdquo Nature Cell Biology vol12 no 9 pp 886ndash893 2010
[16] J Batut B Schmierer J Cao L A Raftery C S Hill and MHowell ldquoTwo highly related regulatory subunits of PP2A exertopposite effects on TGF-120573ActivinNodal signallingrdquo Develop-ment vol 135 no 17 pp 2927ndash2937 2008
[17] A Kurimchak and X Grana ldquoPP2A counterbalances phos-phorylation of pRB and mitotic proteins by multiple CDKs
8 BioMed Research International
potential implications for PP2A disruption in cancerrdquo Genes ampCancer vol 3 no 11-12 pp 739ndash748 2012
[18] G Jayadeva A Kurimchak J Garriga et al ldquoB55120572 PP2A hol-oenzymes modulate the phosphorylation status of the retino-blastomarelated protein p107 and its activationrdquo The Journal ofBiological Chemistry vol 285 no 39 pp 29863ndash29873 2010
[19] J M Overstreet R Samarakoon K K Meldrum and P JHiggins ldquoRedox control of p53 in the transcriptional regulationof TGF-1205731 target genes through SMAD cooperativityrdquo CellularSignalling vol 26 no 7 pp 1427ndash1436 2014
[20] M Motizuki K Isogaya K Miyake et al ldquoOligodendrocytetranscription factor 1 (Olig1) is a smad cofactor involved in cellmotility induced by transforming growth factor-120573rdquoThe Journalof Biological Chemistry vol 288 no 26 pp 18911ndash18922 2013
[21] K Rasanen and A Vaheri ldquoTGF-beta1 causes epithelial-mesenchymal transition in HaCaT derivatives but inducesexpression of COX-2 and migration only in benign not inmalignant keratinocytesrdquo Journal of Dermatological Science vol58 no 2 pp 97ndash104 2010
[22] G Viticchie A M Lena F Cianfarani et al ldquoMicroRNA-203contributes to skin re-epithelializationrdquo Cell Death amp Diseasevol 3 no 11 article e435 2012
[23] Y Yang J Wu H Guan et al ldquoMiR-136 promotes apoptosis ofglioma cells by targeting AEG-1 and Bcl-2rdquo FEBS Letters vol586 no 20 pp 3608ndash3612 2012
[24] D Y Lee Z Jeyapalan L Fang et al ldquoExpression of versican31015840-untranslated region modulates endogenous microrna func-tionsrdquo PLoS ONE vol 5 no 10 Article ID e13599 2010
[25] S Shen H Yue Y Li et al ldquoUpregulation of miR-136 in humannon-small cell lung cancer cells promotes Erk12 activation bytargeting PPP2R2Ardquo Tumor Biology vol 35 no 1 pp 631ndash6402014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
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Diabetes ResearchJournal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
BioMed Research International 3
Table 1 Oligonucleotides for dual-luciferase reporter assays All oligonucleotides for 3 target sites were designed with proper overhangs forSacI and XbaI digestion Mutation sites were shown in italics and KpnI restriction enzyme sites were shown in bold
Name Oligonucleotides sequencewt site 1 sense 51015840-CGGTACCCAACACATTGTTATAGCTACATGGAGAAAGCTT-31015840
wt site 1 antisense 51015840-CTAGAAGCTTTCTCCATGTAGCTATAACAATGTGTTGGGTACCGAGCT-31015840
mut site 1 sense 51015840-CGGTACCCAACACATTGTTATAGCTACGGAACTAAAGCTT-31015840
mut site 1 antisense 51015840-CTAGAAGCTTTAGTTCCGTAGCTATAACAATGTGTTGGGTACCGAGCT-31015840
wt site 2 sense 51015840-CGGTACCACTGAATAAAAGTGACAAAGAATGGAGAATCTGT-31015840
wt site 2 antisense 51015840-CTAGACAGATTCTCCATTCTTTGTCACTTTTATTCAGTGGTACCGAGCT-31015840
mut site 2 sense 51015840-CGGTACCACTGAATAAAAGTGACAAAGCGGAACTAATCTGT-31015840
mut site 2 antisense 51015840-CTAGACAGATTAGTTCCGCTTTGTCACTTTTATTCAGTGGTACCGAGCT-31015840
wt site 3 sense 51015840-CGGTACCGTCAGTCTTGCAGAAACTTTAATGGAGAAGAAAT-31015840
wt site 3 antisense 51015840-CTAGATTTCTTCTCCATTAAAGTTTCTGCAAGACTGACGGTACCGAGCT-31015840
mut site 3 sense 51015840-CGGTACCGTCAGTCTTGCAGAAACTTTCGGAACTAAGAAAT-31015840
mut site 3 antisense 51015840-CTAGATTTCTTAGTTCCGAAAGTTTCTGCAAGACTGACGGTACCGAGCT-31015840
Theoligonucleotide sequences used for each reporter plasmidconstruction are listed in Table 1
27 Western Blot Protein was harvested using RIPA lysisbuffer (Dingguo Beijing China) supplemented with01mmol PMSF diluted with 5 times RSB and denatured at 95∘Cfor 10min Protein lysates were separated by 10 SDS-PAGEand transferred to PVDF membranes the membraneswere blocked in 5 nonfat milk for 1 h and then incubatedwith mouse anti-PPP2R2A (1 1000 CST MA USA) andmouse anti-GAPDH (1 5000 KangChen Shanghai China)antibodies overnight at 4∘C After incubation with HRP-con-jugated secondary antibody (1 5000 KangChen ShanghaiChina) protein bands were visualized using Amersham ECLPrime Western Blotting Detection Reagent (GE HealthcareNJ USA) on Fujifilm LAS-3000 Imager and quantified byImage J
28 Statistical Analysis Data were expressed as the mean plusmnSD from at least three independent experiments Studentrsquos t-test and one-way ANOVA were used to compare the differ-ences between groups All statistical analyses were performedusing SPSS 160 software (SPSS Inc USA) 119875 lt 005 wasconsidered statistically significant
3 Results
31 miR-136 Was Downregulated by TGF-1205731 in HaCaT andNHEKCells To investigate miR-136 expression changes dur-ing proliferation regulation of TGF-1205731 we performed quan-titative real-time PCR assay to measure miR-136 in ker-atinocytes treated with different concentration of TGF-1205731The result showed that the expression level of miR-136 inHaCaT decreased to 019-fold at 48 h after TGF-1205731 treatmentat 2 ngmL and 013-fold for 5 ngmL TGF-1205731 (Figure 1(a))To further confirm this result we isolated and culturedprimary epidermal keratinocytes from normal human skin
The results showed that miR-136 was also significantly down-regulated by TGF-1205731 treatment in NHEK (Figure 1(b))
32 miR-136 Suppression by TGF-1205731 Was Smad3-DependentIt was reported that Smad23 signaling as well as downstreamgene targets such as PAI-1 expression is required for TGF-120573 mediated cytostasis in HaCaT cells [19 20] To investigatewhether canonical Smad23 signaling is required for miR-136 repression during TGF-1205731-induced proliferation arrestin keratinocytes HaCaT cells were transfected with Smad3siRNA for 24 h and then treated with 2 ngmL TGF-1205731 for48 h By quantitative real-time PCR the mRNA level ofSmad3 was decreased to 02-fold at 48 h after transfection(Figure 1(c)) and in Smad3-silenced HaCaT cells TGF-1205731 treatment showed little effect on miR-136 expression(Figure 1(d)) These results indicated that miR-136 was sup-pressed by TGF-1205731 in a Smad3-dependent manner in HaCaTcells
33 Overexpression of miR-136 Overcomes TGF-1205731-InducedProliferation Arrest Previous studies showed that TGF-1205731inhibited cell proliferation in keratinocytes [21] to investigatewhether forced expression of miR-136 is able to modulateTGF-1205731-induced proliferation arrest of keratinocytes HaCaTcells were transfected with miR-136 mimics or miR-NCand subsequently stimulated with 2 ngmL TGF-1205731 for 72 hSuccessful increased expression of miR-136 was confirmedby qRT-PCR (Figure 2(a)) The results of cell proliferationassay showed that TGF-1205731 treatment inhibited HaCaT cellsproliferation effectively and led to gt20 proliferation sup-pression (Figure 2(b)) Moreover transfection of miR-136mimics could negatively regulate the proliferation inhibitionof TGF-1205731 and caused 29 proliferation enhancement Asshown in Figure 2(c) the cell subpopulation in S phasewas obviously decreased in TGF-1205731 treated cells comparedwith the control group while the cell number in S phasewas significantly increased in cells transfected with miR-136mimics versus miR-NC group Taken together these findings
4 BioMed Research International
Control0
02
04
06
08
10
12
HaCaT
2ngmLTGF-1205731
5ngmLTGF-1205731
lowast
lowast
Rela
tive m
iR-136
expr
essio
n
(a)
NHEK
lowast
lowast
Control0
02
04
06
08
10
12
2ngmLTGF-1205731
5ngmLTGF-1205731
Rela
tive m
iR-136
expr
essio
n
(b)
Control0
02
04
06
08
10
12
NC Smad3 siRNA
lowast
Relat
ive S
mad
3 ex
pres
sion
(c)
Smad3 siRNA
Smad3 siRNA
00
02
04
06
08
10
12
TGF-1205731
lowast
lowast
Control+
TGF-1205731+
NC
Rela
tive m
iR-136
expr
essio
n
(d)
Figure 1 miR-136 was downregulated by TGF-1205731 in a Smad3-dependent manner in HaCaT and NHEK cells The relative expression levelsof miR-136 were determined by quantitative real-time PCR (a) The expression of miR-136 was downregulated in HaCaT cells treated with2 ngmLor 5 ngmLTGF-1205731 for 48 h (b)Downregulation ofmiR-136was confirmed inNHEK treatedwithTGF-1205731 at the same concentrationsand time point (c) Smad3 silencing by siRNA was verified by quantitative real-time PCR (d) TGF-1205731-induced miR-136 repression wascounteracted by Smad3 knockdown Data were presented as mean plusmn SD values from three independent experiments Bars indicate SDlowast
119875 lt 005
indicated that miR-136 might act as a modulator of TGF-1205731-induced proliferation arrest in keratinocytes
34 PPP2R2A Was a Direct Target of miR-136 To elucidatethe underlyingmechanisms bywhichmiR-136 exerts its func-tion we exploredmiR-136 targets using the TargetScan bioin-formatics algorithmOur analysis revealed that PPP2R2Awasa potential target of miR-136 based on putative conservedtarget sequences at positions 149ndash155 712ndash719 and 1471ndash1478of the PPP2R2A 31015840-UTR (Figure 3(a)) To further examinewhether miR-136 directly targets PPP2R2A the luciferasereporters containing wild-type or mutant predicted miR-136 binding sites were cotransfected with miR-136 mimicsor NC into Cos-7 cells Luciferase assays were applied 48 hafter transfection and the results showed that compared
to NC transfection with miR-136 resulted in a significantdecrease in renillafirefly luciferase activity of wild-type site1 and site 2 reporter (Figures 3(b) and 3(c)) while therewas no significant decrease of wild-type site 3 reporter(Figure 3(d)) These results suggested that miR-136 repressedPPP2R2A through 2 specific 31015840-UTRbinding sites at positions149ndash155 and 712ndash719 Notably the expression of PPP2R2Ain HaCaT cells substantially decreased at 48 h after miR-136 transfection (Figure 3(e)) Taken together these resultsindicated that miR-136 negatively regulated PPP2R2A in aposttranscriptional manner in HaCaT cells
35 PPP2R2A Was Involved in TGF-1205731-Induced Prolifera-tion Arrest To further establish whether the counterac-tion of miR-136 overexpression against TGF-1205731-induced
BioMed Research International 5
0
5
10
15
20
25
Control miR-136mimics
miR-NC
lowastRe
lativ
e miR
-136
expr
essio
n
(a)
TGF-1205731miR-NCmiR-136
minus
minus
minus
++minus
+minus
minus
+minus
+
0
02
04
06
08
10
12
14 lowast
lowast
OD
val
ue (450
nm)
(b)
0
200
400
600
100
300
500
0 40 80 120 160Control
0
200
400
600
100
300
500
0 40 80 120 1600
200
400
600
0 40 80 120 160miR-NC + TGF-1205731
0
200
400
600
100
300
500
0 40 80 120 160miR-136 + TGF-1205731
S 3817
G0G1 4902G2M 1280 S 1858
G0G1 7424G2M 718
S 1359
G0G1 7958G2M 683 S 2219
G0G1 6676G2M 1105
TGF-1205731
(c)
Figure 2 Overexpression of miR-136 overcame TGF-1205731-induced proliferation arrest HaCaT cells were transfected with miR-136 mimicsor NC (a) The miR-136 level in HaCaT cells transfected with miR-136 mimics for 72 h was verified by qRT-PCR (b) Proliferation assaysin HaCaT cells stimulated with 2 ngmL TGF-1205731 for 24 h (c) Flow cytometry analysis revealed the cell cycle distribution Bars indicate SDlowast
119875 lt 005
proliferation arrest is mediated by repression of PPP2R2Athe expression of PPP2R2A in HaCaT cells treated with2 ngmL TGF-1205731 for 48 h was analyzed by Western blotand the proliferation of PPP2R2A knockdown HaCaT cellsstimulated with TGF-1205731 was assessed by proliferation assayand flow cytometry As shown in Figures 4(a) and 4(b)PPP2R2A in HaCaT cells was upregulated to about 2-foldby 2 ngmL TGF-1205731 treatment for 48 hours and in Figures4(c) and 4(d) knockdown of PPP2R2A clearly counteractedthe proliferation arrest induced by TGF-1205731 In addition thereduction of cell cycle progression at G1S transition inducedby TGF-1205731 was abrogated in PPP2R2A knockdown HaCaTcells The effects of PPP2R2A knockdown were similar tothose induced by miR-136 overexpression Taken togetherthese findings indicated that PPP2R2A was a functionallyimportant target of miR-136 and was involved in the TGF-1205731regulated proliferation of HaCaT cells
4 Discussion
Wound healing is a complex biological process during whichkeratinocyte proliferation and migration are crucial steps forthe rapid closure of the epidermis TGF-1205731 causes wound
margin contraction at the early stage of wound healing andit is responsible for scar formation [22] These processes arecontrolled by a network of biomolecules in a spatiotemporalmanner Several recent reports have indicated that miRNAsare involved in regulating keratinocyte proliferation duringwound healing [10ndash14] Here we aimed to clarify the biolog-ical role of miR-136 in keratinocytes proliferation regulationby TGF-1205731 The experiments showed significant reduction ofmiR-136 in keratinocytes treated with TGF-1205731 and canonicalSmad23 signaling pathway was involved Reintroduction ofmiR-136 by transient transfection as well as silencing bysiRNA of target PPP2R2A blocked TGF-1205731-induced prolif-eration arrest and increased the percentage of keratinocytesin the S phase of the cell cycle while reducing the percentageof those in the G0G1 phase Our results supported thenotion that TGF-1205731-induced proliferation arrest was partiallymediated by miR-136 reduction in HaCaT cells
There are several reports that miR-136 was implicated incell proliferation and played different roles in different typesof cells miR-136 is proposed to be a tumor suppressor inglioma and is capable of targeting the antiapoptosis genesAEG-1 and BCL-2 [23] However miR-136 was found totarget tumor suppressor PTEN in breast cancer cells [24]
6 BioMed Research International
hsa-miR-136
wt site 2mut site 2PtrMmuRnoOcu
hsa-miR-136
wt site 1mut site 1PtrMmuRnoOcu
ACAUUGUUAUAGCUACAUGGAGA
ACAUUGUUAUAGCUACGGAACUA
ACAUUGUUAUAGCUACAUGGAGA
CACAUUUGAUAGCCUCAUGGAGA
CACAUUUGAUAGCCACAUGGAGA
AUUUUUAUAUAGCUACAUGGAGA
1471ndash1478Site 3
712ndash719Site 2
149ndash155Site 1CDS
PPP2R2A
5998400
3998400
5998400
5998400
5998400
59984005998400
5998400
3998400
3998400
3998400
3998400
3998400
3998400
| ||| | |
AGGUAGUAGUUUUGUUUACCUCA
AAAAGUGACAAAG--AAUGGAGA
AAAAGUGACAAAG--CGGAACUA
AAAAGUGACAAAG--AAUGGAGA
AAAAGUGACAAAG--AAUGGAGA
AAAAGUGACAAAG--AAUGGAGA
AAAAGUGACAAAG--AAUGGAGA
5998400
998400 998400
5998400
5998400
5998400
59984005998400
5
3998400
3998400
3998400
3998400
3998400
3998400
| ||||| | | | | |
3 AGGUAGUAGUUUUGUUUACCUCA hsa-miR-136
wt site 3mut site 3PtrMmuRnoOcu
UCUUGCAGAAACUUUAAUGGAGA
UCUUGCAGAAACUUUCGGAACUA
UCUUGCAGAAACUUUAAUGGAGA
GUCCUGCGGAAGCUUUAUGGAGA
GUCCUGCGAAAGCUUUAUGGAGA
GUCUUGAAGAGCUUUAAUGGAGA
5998400
998400 998400
5998400
5998400
5998400
59984005998400
5
3998400399840039984003998400
39984003998400
||| || ||
3 AGGUAGUAGUUUUGUUUACCUCA
(a)
0020406081012
miR-NCmiR-136
+minus
+minus
minus
+minus
+wt site 1 mut site 1
lowast
Rela
tive l
ucife
rase
activ
ity
(b)
0020406081012
miR-NCmiR-136
+minus
+minus
minus
+minus
+wt site 2 mut site 2
lowastRe
lativ
e luc
ifera
se ac
tivity
(c)
0020406081012
miR-NCmiR-136
+minus
+minus
minus
+minus
+wt site 3 mut site 3
NS
Relat
ive l
ucife
rase
activ
ity
(d)
PPP2R2A
GAPDH
miR-136miR-NC mimics
(e)
Figure 3 PPP2R2A was a direct target of miR-136 (a) There were three potential miR-136 binding sites in PPP2R2A 31015840-UTR based on theTargetScan database the conservation of the miR-136 binding seed regions among different species was shown in shading and mutationswere shown in italics Fragments containing wild-type (wt) or mutant (mut) miR-136 binding sites in human PPP2R2A 31015840-UTR were cloneddownstreamof the luciferase reporter gene separately ((b)ndash(d)) Luciferase reporter assay (119899 = 3 for each group) Cos-7 cellswere cotransfectedwith a 31015840-UTR reporter construct and the miR-136 mimics or miR-NC and the results showed that site 1 and site 2 were the direct targets ofmiR-136 Luciferase activityrenilla activity was applied as the baseline control for the experiments using the same reporter Data representmean plusmn SD lowast119875 lt 005 (e) Western blot analyses of PPP2R2A expression in HaCaT cells transfected with miR-136 mimics or miR-NCGAPDH was used as loading control
PPP2R2A
GAPDH
Control TGF-1205731
(a)
0005101520253035 lowast
Control TGF-1205731Relat
ive P
PP2R
2A ex
pres
sion
(b)
Control NC PPP2R2A00020406081012 lowast
siRNA
Relat
ive P
PP2R
2A ex
pres
sion
(c)
Control0
02040608101214
TGF-1205731 +
lowast
TGF-1205731+
NC
OD
val
ue (450
nm)
PPP2R2A siRNA
(d)
Control
0
200
400
600
100
300
500
0 40 80 120 160
S 3820
G0G1 4906G2M 1274
0 40 80 120 1600
200
400
600
TGF-1205731 + NC
S 1367
G0G17889G2M 743
0
200
400
600
0 40 80 120 160TGF-1205731 +
PPP2R2A siRNA
S 2156
G0G1 6693G2M 1150
(e)
Figure 4 PPP2R2A was involved in TGF-1205731-induced proliferation arrest in HaCaT cells (a) Western blot analysis of PPP2R2A expression inresponse to TGF-1205731 treatment GAPDHwas used as internal control (b)Three independent results ofWestern blot were quantified by ImageJ (c) PPP2R2A silencing was verified by quantitative real-time PCR (d) Determination of cell viability with Cell Counting Kit-8 (e) Flowcytometry analysis of the cell cycle Bars indicate SD lowast119875 lt 005
BioMed Research International 7
TGF-1205731 PPP2R2AmiR-136
Keratinocytes proliferation
Figure 5 Schematic representation summarizing the roles of miR-136 and PPP2R2A during TGF-1205731-induced proliferation arrest Seetext for details
Recently results of others indicated that miR-136 enhancedphosphorylation of Erk12 through inhibition of PPP2R2Aexpression to promoted cell proliferation in human non-small cell lung cancer and the sequence at position 149ndash155 ofthe PPP2R2A 31015840-UTR was determined to be the target site ofmiR-136 [25] In this study we clarified miR-136 suppressedPPP2R2A expression by directly targeting two conservedregions at positions 149ndash155 and 712ndash719 in its 31015840-UTRFurthermore the protein level of PPP2R2A was significantlyreduced by miR-136 mimics transfection in HaCaT cellsTherefore we proposed that in the presence of TGF-1205731 miR-136 was a positive modulator of keratinocytes proliferationand PPP2R2A was a functional target of miR-136 via bindingto its 31015840-UTR region
In conclusion our results indicated that the miR-136treatment bypassed TGF-1205731-induced proliferation arrest atleast partially through downregulation of PPP2R2A expres-sion (Figure 5) Our data suggested that modulation ofkeratinocytes proliferation by miR-136 depends on the cel-lular environment especially in the presence of TGF-1205731which likely has major effects on the expression of miR-136target gene PPP2R2A Further studies are needed to deter-mine whether miR-136 based therapeutic strategies could beexploited to protect keratinocytes proliferation and functionin the condition of enhanced TGF-1205731 during wound healing
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Dianbao Zhang and Jing Wang contributed equally to thiswork
Acknowledgments
This work was funded by the National Basic ResearchProgram of China (2012CB518103) National Natural ScienceFoundation of China (81370883) Doctoral Fund of Ministryof Education of China (20132104110020) Science and Tech-nology Planning Project of Liaoning Province (2012225080)and Science and Technology Planning Project of Shenyang(F11-262-9-01) The authors thank Dr Hongchuan Xin andDr Ziwei Miao for critical reading and editing of this paper
References
[1] I Haase R Evans R Pofahl and F M Watt ldquoRegulation ofkeratinocyte shape migration and wound epithelialization byIGF-1 and EGF-dependent signalling pathwaysrdquo Journal of CellScience vol 116 no 15 pp 3227ndash3238 2003
[2] E Fuchs ldquoSkin stem cells rising to the surfacerdquo Journal of CellBiology vol 180 no 2 pp 273ndash284 2008
[3] C Pincelli andAMarconi ldquoKeratinocyte stem cells friends andfoesrdquo Journal of Cellular Physiology vol 225 no 2 pp 310ndash3152010
[4] J Nie X Fu and W Han ldquoMicroenvironment-dependenthomeostasis and differentiation of epidermal basal undiffer-entiated keratinocytes and their clinical applications in skinrepairrdquo Journal of the European Academy of Dermatology andVenereology vol 27 no 5 pp 531ndash535 2013
[5] S Guo and L A Dipietro ldquoFactors affecting wound healingrdquoJournal of Dental Research vol 89 no 3 pp 219ndash229 2010
[6] G S Schultz and AWysocki ldquoInteractions between extracellu-lar matrix and growth factors in wound healingrdquoWound Repairand Regeneration vol 17 no 2 pp 153ndash162 2009
[7] J S Kang C Liu andRDerynck ldquoNew regulatorymechanismsof TGF-120573 receptor functionrdquo Trends in Cell Biology vol 19 no8 pp 385ndash394 2009
[8] R Yi and E Fuchs ldquoMicroRNA-mediated control in the skinrdquoCell Death amp Differentiation vol 17 no 2 pp 229ndash235 2010
[9] M Teta Y S Choi T Okegbe et al ldquoInducible deletion of epi-dermal Dicer and Drosha reveals multiple functions for miR-NAs in postnatal skinrdquo Development vol 139 no 8 pp 1405ndash1416 2012
[10] D AntoniniM T Russo L de RosaMGorrese L del Vecchioand C Missero ldquoTranscriptional repression of miR-34 familycontributes to p63-mediated cell cycle progression in epidermalcellsrdquo Journal of Investigative Dermatology vol 130 no 5 pp1249ndash1257 2010
[11] N Xu P Brodin T Wei et al ldquoMiR-125b a MicroRNA down-regulated in psoriasis modulates keratinocyte proliferation bytargeting FGFR2rdquo Journal of Investigative Dermatology vol 131no 7 pp 1521ndash1529 2011
[12] Z Lin X Wang C Fewell J Cameron Q Yin and E KFlemington ldquoDifferential expression of the miR-200 familymicroRNAs in epithelial and B cells and regulation of Epstein-Barr virus reactivation by the miR-200 family member miR-429rdquo Journal of Virology vol 84 no 15 pp 7892ndash7897 2010
[13] A M Lena R Shalom-Feuerstein P R di Val Cervo et alldquomiR-203 represses ldquostemnessrdquo by repressingΔNp63rdquoCellDeathamp Differentiation vol 15 no 7 pp 1187ndash1195 2008
[14] S Biswas S Roy J Banerjee et al ldquoHypoxia inducible microR-NA 210 attenuates keratinocyte proliferation and impairs clo-sure in a murine model of ischemic woundsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 107 no 15 pp 6976ndash6981 2010
[15] M H Schmitz M Held V Janssens et al ldquoLive-cell imagingRNAi screen identifies PP2A-B55120572 and importin-120573 21 as keymitotic exit regulators in human cellsrdquo Nature Cell Biology vol12 no 9 pp 886ndash893 2010
[16] J Batut B Schmierer J Cao L A Raftery C S Hill and MHowell ldquoTwo highly related regulatory subunits of PP2A exertopposite effects on TGF-120573ActivinNodal signallingrdquo Develop-ment vol 135 no 17 pp 2927ndash2937 2008
[17] A Kurimchak and X Grana ldquoPP2A counterbalances phos-phorylation of pRB and mitotic proteins by multiple CDKs
8 BioMed Research International
potential implications for PP2A disruption in cancerrdquo Genes ampCancer vol 3 no 11-12 pp 739ndash748 2012
[18] G Jayadeva A Kurimchak J Garriga et al ldquoB55120572 PP2A hol-oenzymes modulate the phosphorylation status of the retino-blastomarelated protein p107 and its activationrdquo The Journal ofBiological Chemistry vol 285 no 39 pp 29863ndash29873 2010
[19] J M Overstreet R Samarakoon K K Meldrum and P JHiggins ldquoRedox control of p53 in the transcriptional regulationof TGF-1205731 target genes through SMAD cooperativityrdquo CellularSignalling vol 26 no 7 pp 1427ndash1436 2014
[20] M Motizuki K Isogaya K Miyake et al ldquoOligodendrocytetranscription factor 1 (Olig1) is a smad cofactor involved in cellmotility induced by transforming growth factor-120573rdquoThe Journalof Biological Chemistry vol 288 no 26 pp 18911ndash18922 2013
[21] K Rasanen and A Vaheri ldquoTGF-beta1 causes epithelial-mesenchymal transition in HaCaT derivatives but inducesexpression of COX-2 and migration only in benign not inmalignant keratinocytesrdquo Journal of Dermatological Science vol58 no 2 pp 97ndash104 2010
[22] G Viticchie A M Lena F Cianfarani et al ldquoMicroRNA-203contributes to skin re-epithelializationrdquo Cell Death amp Diseasevol 3 no 11 article e435 2012
[23] Y Yang J Wu H Guan et al ldquoMiR-136 promotes apoptosis ofglioma cells by targeting AEG-1 and Bcl-2rdquo FEBS Letters vol586 no 20 pp 3608ndash3612 2012
[24] D Y Lee Z Jeyapalan L Fang et al ldquoExpression of versican31015840-untranslated region modulates endogenous microrna func-tionsrdquo PLoS ONE vol 5 no 10 Article ID e13599 2010
[25] S Shen H Yue Y Li et al ldquoUpregulation of miR-136 in humannon-small cell lung cancer cells promotes Erk12 activation bytargeting PPP2R2Ardquo Tumor Biology vol 35 no 1 pp 631ndash6402014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
4 BioMed Research International
Control0
02
04
06
08
10
12
HaCaT
2ngmLTGF-1205731
5ngmLTGF-1205731
lowast
lowast
Rela
tive m
iR-136
expr
essio
n
(a)
NHEK
lowast
lowast
Control0
02
04
06
08
10
12
2ngmLTGF-1205731
5ngmLTGF-1205731
Rela
tive m
iR-136
expr
essio
n
(b)
Control0
02
04
06
08
10
12
NC Smad3 siRNA
lowast
Relat
ive S
mad
3 ex
pres
sion
(c)
Smad3 siRNA
Smad3 siRNA
00
02
04
06
08
10
12
TGF-1205731
lowast
lowast
Control+
TGF-1205731+
NC
Rela
tive m
iR-136
expr
essio
n
(d)
Figure 1 miR-136 was downregulated by TGF-1205731 in a Smad3-dependent manner in HaCaT and NHEK cells The relative expression levelsof miR-136 were determined by quantitative real-time PCR (a) The expression of miR-136 was downregulated in HaCaT cells treated with2 ngmLor 5 ngmLTGF-1205731 for 48 h (b)Downregulation ofmiR-136was confirmed inNHEK treatedwithTGF-1205731 at the same concentrationsand time point (c) Smad3 silencing by siRNA was verified by quantitative real-time PCR (d) TGF-1205731-induced miR-136 repression wascounteracted by Smad3 knockdown Data were presented as mean plusmn SD values from three independent experiments Bars indicate SDlowast
119875 lt 005
indicated that miR-136 might act as a modulator of TGF-1205731-induced proliferation arrest in keratinocytes
34 PPP2R2A Was a Direct Target of miR-136 To elucidatethe underlyingmechanisms bywhichmiR-136 exerts its func-tion we exploredmiR-136 targets using the TargetScan bioin-formatics algorithmOur analysis revealed that PPP2R2Awasa potential target of miR-136 based on putative conservedtarget sequences at positions 149ndash155 712ndash719 and 1471ndash1478of the PPP2R2A 31015840-UTR (Figure 3(a)) To further examinewhether miR-136 directly targets PPP2R2A the luciferasereporters containing wild-type or mutant predicted miR-136 binding sites were cotransfected with miR-136 mimicsor NC into Cos-7 cells Luciferase assays were applied 48 hafter transfection and the results showed that compared
to NC transfection with miR-136 resulted in a significantdecrease in renillafirefly luciferase activity of wild-type site1 and site 2 reporter (Figures 3(b) and 3(c)) while therewas no significant decrease of wild-type site 3 reporter(Figure 3(d)) These results suggested that miR-136 repressedPPP2R2A through 2 specific 31015840-UTRbinding sites at positions149ndash155 and 712ndash719 Notably the expression of PPP2R2Ain HaCaT cells substantially decreased at 48 h after miR-136 transfection (Figure 3(e)) Taken together these resultsindicated that miR-136 negatively regulated PPP2R2A in aposttranscriptional manner in HaCaT cells
35 PPP2R2A Was Involved in TGF-1205731-Induced Prolifera-tion Arrest To further establish whether the counterac-tion of miR-136 overexpression against TGF-1205731-induced
BioMed Research International 5
0
5
10
15
20
25
Control miR-136mimics
miR-NC
lowastRe
lativ
e miR
-136
expr
essio
n
(a)
TGF-1205731miR-NCmiR-136
minus
minus
minus
++minus
+minus
minus
+minus
+
0
02
04
06
08
10
12
14 lowast
lowast
OD
val
ue (450
nm)
(b)
0
200
400
600
100
300
500
0 40 80 120 160Control
0
200
400
600
100
300
500
0 40 80 120 1600
200
400
600
0 40 80 120 160miR-NC + TGF-1205731
0
200
400
600
100
300
500
0 40 80 120 160miR-136 + TGF-1205731
S 3817
G0G1 4902G2M 1280 S 1858
G0G1 7424G2M 718
S 1359
G0G1 7958G2M 683 S 2219
G0G1 6676G2M 1105
TGF-1205731
(c)
Figure 2 Overexpression of miR-136 overcame TGF-1205731-induced proliferation arrest HaCaT cells were transfected with miR-136 mimicsor NC (a) The miR-136 level in HaCaT cells transfected with miR-136 mimics for 72 h was verified by qRT-PCR (b) Proliferation assaysin HaCaT cells stimulated with 2 ngmL TGF-1205731 for 24 h (c) Flow cytometry analysis revealed the cell cycle distribution Bars indicate SDlowast
119875 lt 005
proliferation arrest is mediated by repression of PPP2R2Athe expression of PPP2R2A in HaCaT cells treated with2 ngmL TGF-1205731 for 48 h was analyzed by Western blotand the proliferation of PPP2R2A knockdown HaCaT cellsstimulated with TGF-1205731 was assessed by proliferation assayand flow cytometry As shown in Figures 4(a) and 4(b)PPP2R2A in HaCaT cells was upregulated to about 2-foldby 2 ngmL TGF-1205731 treatment for 48 hours and in Figures4(c) and 4(d) knockdown of PPP2R2A clearly counteractedthe proliferation arrest induced by TGF-1205731 In addition thereduction of cell cycle progression at G1S transition inducedby TGF-1205731 was abrogated in PPP2R2A knockdown HaCaTcells The effects of PPP2R2A knockdown were similar tothose induced by miR-136 overexpression Taken togetherthese findings indicated that PPP2R2A was a functionallyimportant target of miR-136 and was involved in the TGF-1205731regulated proliferation of HaCaT cells
4 Discussion
Wound healing is a complex biological process during whichkeratinocyte proliferation and migration are crucial steps forthe rapid closure of the epidermis TGF-1205731 causes wound
margin contraction at the early stage of wound healing andit is responsible for scar formation [22] These processes arecontrolled by a network of biomolecules in a spatiotemporalmanner Several recent reports have indicated that miRNAsare involved in regulating keratinocyte proliferation duringwound healing [10ndash14] Here we aimed to clarify the biolog-ical role of miR-136 in keratinocytes proliferation regulationby TGF-1205731 The experiments showed significant reduction ofmiR-136 in keratinocytes treated with TGF-1205731 and canonicalSmad23 signaling pathway was involved Reintroduction ofmiR-136 by transient transfection as well as silencing bysiRNA of target PPP2R2A blocked TGF-1205731-induced prolif-eration arrest and increased the percentage of keratinocytesin the S phase of the cell cycle while reducing the percentageof those in the G0G1 phase Our results supported thenotion that TGF-1205731-induced proliferation arrest was partiallymediated by miR-136 reduction in HaCaT cells
There are several reports that miR-136 was implicated incell proliferation and played different roles in different typesof cells miR-136 is proposed to be a tumor suppressor inglioma and is capable of targeting the antiapoptosis genesAEG-1 and BCL-2 [23] However miR-136 was found totarget tumor suppressor PTEN in breast cancer cells [24]
6 BioMed Research International
hsa-miR-136
wt site 2mut site 2PtrMmuRnoOcu
hsa-miR-136
wt site 1mut site 1PtrMmuRnoOcu
ACAUUGUUAUAGCUACAUGGAGA
ACAUUGUUAUAGCUACGGAACUA
ACAUUGUUAUAGCUACAUGGAGA
CACAUUUGAUAGCCUCAUGGAGA
CACAUUUGAUAGCCACAUGGAGA
AUUUUUAUAUAGCUACAUGGAGA
1471ndash1478Site 3
712ndash719Site 2
149ndash155Site 1CDS
PPP2R2A
5998400
3998400
5998400
5998400
5998400
59984005998400
5998400
3998400
3998400
3998400
3998400
3998400
3998400
| ||| | |
AGGUAGUAGUUUUGUUUACCUCA
AAAAGUGACAAAG--AAUGGAGA
AAAAGUGACAAAG--CGGAACUA
AAAAGUGACAAAG--AAUGGAGA
AAAAGUGACAAAG--AAUGGAGA
AAAAGUGACAAAG--AAUGGAGA
AAAAGUGACAAAG--AAUGGAGA
5998400
998400 998400
5998400
5998400
5998400
59984005998400
5
3998400
3998400
3998400
3998400
3998400
3998400
| ||||| | | | | |
3 AGGUAGUAGUUUUGUUUACCUCA hsa-miR-136
wt site 3mut site 3PtrMmuRnoOcu
UCUUGCAGAAACUUUAAUGGAGA
UCUUGCAGAAACUUUCGGAACUA
UCUUGCAGAAACUUUAAUGGAGA
GUCCUGCGGAAGCUUUAUGGAGA
GUCCUGCGAAAGCUUUAUGGAGA
GUCUUGAAGAGCUUUAAUGGAGA
5998400
998400 998400
5998400
5998400
5998400
59984005998400
5
3998400399840039984003998400
39984003998400
||| || ||
3 AGGUAGUAGUUUUGUUUACCUCA
(a)
0020406081012
miR-NCmiR-136
+minus
+minus
minus
+minus
+wt site 1 mut site 1
lowast
Rela
tive l
ucife
rase
activ
ity
(b)
0020406081012
miR-NCmiR-136
+minus
+minus
minus
+minus
+wt site 2 mut site 2
lowastRe
lativ
e luc
ifera
se ac
tivity
(c)
0020406081012
miR-NCmiR-136
+minus
+minus
minus
+minus
+wt site 3 mut site 3
NS
Relat
ive l
ucife
rase
activ
ity
(d)
PPP2R2A
GAPDH
miR-136miR-NC mimics
(e)
Figure 3 PPP2R2A was a direct target of miR-136 (a) There were three potential miR-136 binding sites in PPP2R2A 31015840-UTR based on theTargetScan database the conservation of the miR-136 binding seed regions among different species was shown in shading and mutationswere shown in italics Fragments containing wild-type (wt) or mutant (mut) miR-136 binding sites in human PPP2R2A 31015840-UTR were cloneddownstreamof the luciferase reporter gene separately ((b)ndash(d)) Luciferase reporter assay (119899 = 3 for each group) Cos-7 cellswere cotransfectedwith a 31015840-UTR reporter construct and the miR-136 mimics or miR-NC and the results showed that site 1 and site 2 were the direct targets ofmiR-136 Luciferase activityrenilla activity was applied as the baseline control for the experiments using the same reporter Data representmean plusmn SD lowast119875 lt 005 (e) Western blot analyses of PPP2R2A expression in HaCaT cells transfected with miR-136 mimics or miR-NCGAPDH was used as loading control
PPP2R2A
GAPDH
Control TGF-1205731
(a)
0005101520253035 lowast
Control TGF-1205731Relat
ive P
PP2R
2A ex
pres
sion
(b)
Control NC PPP2R2A00020406081012 lowast
siRNA
Relat
ive P
PP2R
2A ex
pres
sion
(c)
Control0
02040608101214
TGF-1205731 +
lowast
TGF-1205731+
NC
OD
val
ue (450
nm)
PPP2R2A siRNA
(d)
Control
0
200
400
600
100
300
500
0 40 80 120 160
S 3820
G0G1 4906G2M 1274
0 40 80 120 1600
200
400
600
TGF-1205731 + NC
S 1367
G0G17889G2M 743
0
200
400
600
0 40 80 120 160TGF-1205731 +
PPP2R2A siRNA
S 2156
G0G1 6693G2M 1150
(e)
Figure 4 PPP2R2A was involved in TGF-1205731-induced proliferation arrest in HaCaT cells (a) Western blot analysis of PPP2R2A expression inresponse to TGF-1205731 treatment GAPDHwas used as internal control (b)Three independent results ofWestern blot were quantified by ImageJ (c) PPP2R2A silencing was verified by quantitative real-time PCR (d) Determination of cell viability with Cell Counting Kit-8 (e) Flowcytometry analysis of the cell cycle Bars indicate SD lowast119875 lt 005
BioMed Research International 7
TGF-1205731 PPP2R2AmiR-136
Keratinocytes proliferation
Figure 5 Schematic representation summarizing the roles of miR-136 and PPP2R2A during TGF-1205731-induced proliferation arrest Seetext for details
Recently results of others indicated that miR-136 enhancedphosphorylation of Erk12 through inhibition of PPP2R2Aexpression to promoted cell proliferation in human non-small cell lung cancer and the sequence at position 149ndash155 ofthe PPP2R2A 31015840-UTR was determined to be the target site ofmiR-136 [25] In this study we clarified miR-136 suppressedPPP2R2A expression by directly targeting two conservedregions at positions 149ndash155 and 712ndash719 in its 31015840-UTRFurthermore the protein level of PPP2R2A was significantlyreduced by miR-136 mimics transfection in HaCaT cellsTherefore we proposed that in the presence of TGF-1205731 miR-136 was a positive modulator of keratinocytes proliferationand PPP2R2A was a functional target of miR-136 via bindingto its 31015840-UTR region
In conclusion our results indicated that the miR-136treatment bypassed TGF-1205731-induced proliferation arrest atleast partially through downregulation of PPP2R2A expres-sion (Figure 5) Our data suggested that modulation ofkeratinocytes proliferation by miR-136 depends on the cel-lular environment especially in the presence of TGF-1205731which likely has major effects on the expression of miR-136target gene PPP2R2A Further studies are needed to deter-mine whether miR-136 based therapeutic strategies could beexploited to protect keratinocytes proliferation and functionin the condition of enhanced TGF-1205731 during wound healing
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Dianbao Zhang and Jing Wang contributed equally to thiswork
Acknowledgments
This work was funded by the National Basic ResearchProgram of China (2012CB518103) National Natural ScienceFoundation of China (81370883) Doctoral Fund of Ministryof Education of China (20132104110020) Science and Tech-nology Planning Project of Liaoning Province (2012225080)and Science and Technology Planning Project of Shenyang(F11-262-9-01) The authors thank Dr Hongchuan Xin andDr Ziwei Miao for critical reading and editing of this paper
References
[1] I Haase R Evans R Pofahl and F M Watt ldquoRegulation ofkeratinocyte shape migration and wound epithelialization byIGF-1 and EGF-dependent signalling pathwaysrdquo Journal of CellScience vol 116 no 15 pp 3227ndash3238 2003
[2] E Fuchs ldquoSkin stem cells rising to the surfacerdquo Journal of CellBiology vol 180 no 2 pp 273ndash284 2008
[3] C Pincelli andAMarconi ldquoKeratinocyte stem cells friends andfoesrdquo Journal of Cellular Physiology vol 225 no 2 pp 310ndash3152010
[4] J Nie X Fu and W Han ldquoMicroenvironment-dependenthomeostasis and differentiation of epidermal basal undiffer-entiated keratinocytes and their clinical applications in skinrepairrdquo Journal of the European Academy of Dermatology andVenereology vol 27 no 5 pp 531ndash535 2013
[5] S Guo and L A Dipietro ldquoFactors affecting wound healingrdquoJournal of Dental Research vol 89 no 3 pp 219ndash229 2010
[6] G S Schultz and AWysocki ldquoInteractions between extracellu-lar matrix and growth factors in wound healingrdquoWound Repairand Regeneration vol 17 no 2 pp 153ndash162 2009
[7] J S Kang C Liu andRDerynck ldquoNew regulatorymechanismsof TGF-120573 receptor functionrdquo Trends in Cell Biology vol 19 no8 pp 385ndash394 2009
[8] R Yi and E Fuchs ldquoMicroRNA-mediated control in the skinrdquoCell Death amp Differentiation vol 17 no 2 pp 229ndash235 2010
[9] M Teta Y S Choi T Okegbe et al ldquoInducible deletion of epi-dermal Dicer and Drosha reveals multiple functions for miR-NAs in postnatal skinrdquo Development vol 139 no 8 pp 1405ndash1416 2012
[10] D AntoniniM T Russo L de RosaMGorrese L del Vecchioand C Missero ldquoTranscriptional repression of miR-34 familycontributes to p63-mediated cell cycle progression in epidermalcellsrdquo Journal of Investigative Dermatology vol 130 no 5 pp1249ndash1257 2010
[11] N Xu P Brodin T Wei et al ldquoMiR-125b a MicroRNA down-regulated in psoriasis modulates keratinocyte proliferation bytargeting FGFR2rdquo Journal of Investigative Dermatology vol 131no 7 pp 1521ndash1529 2011
[12] Z Lin X Wang C Fewell J Cameron Q Yin and E KFlemington ldquoDifferential expression of the miR-200 familymicroRNAs in epithelial and B cells and regulation of Epstein-Barr virus reactivation by the miR-200 family member miR-429rdquo Journal of Virology vol 84 no 15 pp 7892ndash7897 2010
[13] A M Lena R Shalom-Feuerstein P R di Val Cervo et alldquomiR-203 represses ldquostemnessrdquo by repressingΔNp63rdquoCellDeathamp Differentiation vol 15 no 7 pp 1187ndash1195 2008
[14] S Biswas S Roy J Banerjee et al ldquoHypoxia inducible microR-NA 210 attenuates keratinocyte proliferation and impairs clo-sure in a murine model of ischemic woundsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 107 no 15 pp 6976ndash6981 2010
[15] M H Schmitz M Held V Janssens et al ldquoLive-cell imagingRNAi screen identifies PP2A-B55120572 and importin-120573 21 as keymitotic exit regulators in human cellsrdquo Nature Cell Biology vol12 no 9 pp 886ndash893 2010
[16] J Batut B Schmierer J Cao L A Raftery C S Hill and MHowell ldquoTwo highly related regulatory subunits of PP2A exertopposite effects on TGF-120573ActivinNodal signallingrdquo Develop-ment vol 135 no 17 pp 2927ndash2937 2008
[17] A Kurimchak and X Grana ldquoPP2A counterbalances phos-phorylation of pRB and mitotic proteins by multiple CDKs
8 BioMed Research International
potential implications for PP2A disruption in cancerrdquo Genes ampCancer vol 3 no 11-12 pp 739ndash748 2012
[18] G Jayadeva A Kurimchak J Garriga et al ldquoB55120572 PP2A hol-oenzymes modulate the phosphorylation status of the retino-blastomarelated protein p107 and its activationrdquo The Journal ofBiological Chemistry vol 285 no 39 pp 29863ndash29873 2010
[19] J M Overstreet R Samarakoon K K Meldrum and P JHiggins ldquoRedox control of p53 in the transcriptional regulationof TGF-1205731 target genes through SMAD cooperativityrdquo CellularSignalling vol 26 no 7 pp 1427ndash1436 2014
[20] M Motizuki K Isogaya K Miyake et al ldquoOligodendrocytetranscription factor 1 (Olig1) is a smad cofactor involved in cellmotility induced by transforming growth factor-120573rdquoThe Journalof Biological Chemistry vol 288 no 26 pp 18911ndash18922 2013
[21] K Rasanen and A Vaheri ldquoTGF-beta1 causes epithelial-mesenchymal transition in HaCaT derivatives but inducesexpression of COX-2 and migration only in benign not inmalignant keratinocytesrdquo Journal of Dermatological Science vol58 no 2 pp 97ndash104 2010
[22] G Viticchie A M Lena F Cianfarani et al ldquoMicroRNA-203contributes to skin re-epithelializationrdquo Cell Death amp Diseasevol 3 no 11 article e435 2012
[23] Y Yang J Wu H Guan et al ldquoMiR-136 promotes apoptosis ofglioma cells by targeting AEG-1 and Bcl-2rdquo FEBS Letters vol586 no 20 pp 3608ndash3612 2012
[24] D Y Lee Z Jeyapalan L Fang et al ldquoExpression of versican31015840-untranslated region modulates endogenous microrna func-tionsrdquo PLoS ONE vol 5 no 10 Article ID e13599 2010
[25] S Shen H Yue Y Li et al ldquoUpregulation of miR-136 in humannon-small cell lung cancer cells promotes Erk12 activation bytargeting PPP2R2Ardquo Tumor Biology vol 35 no 1 pp 631ndash6402014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
BioMed Research International 5
0
5
10
15
20
25
Control miR-136mimics
miR-NC
lowastRe
lativ
e miR
-136
expr
essio
n
(a)
TGF-1205731miR-NCmiR-136
minus
minus
minus
++minus
+minus
minus
+minus
+
0
02
04
06
08
10
12
14 lowast
lowast
OD
val
ue (450
nm)
(b)
0
200
400
600
100
300
500
0 40 80 120 160Control
0
200
400
600
100
300
500
0 40 80 120 1600
200
400
600
0 40 80 120 160miR-NC + TGF-1205731
0
200
400
600
100
300
500
0 40 80 120 160miR-136 + TGF-1205731
S 3817
G0G1 4902G2M 1280 S 1858
G0G1 7424G2M 718
S 1359
G0G1 7958G2M 683 S 2219
G0G1 6676G2M 1105
TGF-1205731
(c)
Figure 2 Overexpression of miR-136 overcame TGF-1205731-induced proliferation arrest HaCaT cells were transfected with miR-136 mimicsor NC (a) The miR-136 level in HaCaT cells transfected with miR-136 mimics for 72 h was verified by qRT-PCR (b) Proliferation assaysin HaCaT cells stimulated with 2 ngmL TGF-1205731 for 24 h (c) Flow cytometry analysis revealed the cell cycle distribution Bars indicate SDlowast
119875 lt 005
proliferation arrest is mediated by repression of PPP2R2Athe expression of PPP2R2A in HaCaT cells treated with2 ngmL TGF-1205731 for 48 h was analyzed by Western blotand the proliferation of PPP2R2A knockdown HaCaT cellsstimulated with TGF-1205731 was assessed by proliferation assayand flow cytometry As shown in Figures 4(a) and 4(b)PPP2R2A in HaCaT cells was upregulated to about 2-foldby 2 ngmL TGF-1205731 treatment for 48 hours and in Figures4(c) and 4(d) knockdown of PPP2R2A clearly counteractedthe proliferation arrest induced by TGF-1205731 In addition thereduction of cell cycle progression at G1S transition inducedby TGF-1205731 was abrogated in PPP2R2A knockdown HaCaTcells The effects of PPP2R2A knockdown were similar tothose induced by miR-136 overexpression Taken togetherthese findings indicated that PPP2R2A was a functionallyimportant target of miR-136 and was involved in the TGF-1205731regulated proliferation of HaCaT cells
4 Discussion
Wound healing is a complex biological process during whichkeratinocyte proliferation and migration are crucial steps forthe rapid closure of the epidermis TGF-1205731 causes wound
margin contraction at the early stage of wound healing andit is responsible for scar formation [22] These processes arecontrolled by a network of biomolecules in a spatiotemporalmanner Several recent reports have indicated that miRNAsare involved in regulating keratinocyte proliferation duringwound healing [10ndash14] Here we aimed to clarify the biolog-ical role of miR-136 in keratinocytes proliferation regulationby TGF-1205731 The experiments showed significant reduction ofmiR-136 in keratinocytes treated with TGF-1205731 and canonicalSmad23 signaling pathway was involved Reintroduction ofmiR-136 by transient transfection as well as silencing bysiRNA of target PPP2R2A blocked TGF-1205731-induced prolif-eration arrest and increased the percentage of keratinocytesin the S phase of the cell cycle while reducing the percentageof those in the G0G1 phase Our results supported thenotion that TGF-1205731-induced proliferation arrest was partiallymediated by miR-136 reduction in HaCaT cells
There are several reports that miR-136 was implicated incell proliferation and played different roles in different typesof cells miR-136 is proposed to be a tumor suppressor inglioma and is capable of targeting the antiapoptosis genesAEG-1 and BCL-2 [23] However miR-136 was found totarget tumor suppressor PTEN in breast cancer cells [24]
6 BioMed Research International
hsa-miR-136
wt site 2mut site 2PtrMmuRnoOcu
hsa-miR-136
wt site 1mut site 1PtrMmuRnoOcu
ACAUUGUUAUAGCUACAUGGAGA
ACAUUGUUAUAGCUACGGAACUA
ACAUUGUUAUAGCUACAUGGAGA
CACAUUUGAUAGCCUCAUGGAGA
CACAUUUGAUAGCCACAUGGAGA
AUUUUUAUAUAGCUACAUGGAGA
1471ndash1478Site 3
712ndash719Site 2
149ndash155Site 1CDS
PPP2R2A
5998400
3998400
5998400
5998400
5998400
59984005998400
5998400
3998400
3998400
3998400
3998400
3998400
3998400
| ||| | |
AGGUAGUAGUUUUGUUUACCUCA
AAAAGUGACAAAG--AAUGGAGA
AAAAGUGACAAAG--CGGAACUA
AAAAGUGACAAAG--AAUGGAGA
AAAAGUGACAAAG--AAUGGAGA
AAAAGUGACAAAG--AAUGGAGA
AAAAGUGACAAAG--AAUGGAGA
5998400
998400 998400
5998400
5998400
5998400
59984005998400
5
3998400
3998400
3998400
3998400
3998400
3998400
| ||||| | | | | |
3 AGGUAGUAGUUUUGUUUACCUCA hsa-miR-136
wt site 3mut site 3PtrMmuRnoOcu
UCUUGCAGAAACUUUAAUGGAGA
UCUUGCAGAAACUUUCGGAACUA
UCUUGCAGAAACUUUAAUGGAGA
GUCCUGCGGAAGCUUUAUGGAGA
GUCCUGCGAAAGCUUUAUGGAGA
GUCUUGAAGAGCUUUAAUGGAGA
5998400
998400 998400
5998400
5998400
5998400
59984005998400
5
3998400399840039984003998400
39984003998400
||| || ||
3 AGGUAGUAGUUUUGUUUACCUCA
(a)
0020406081012
miR-NCmiR-136
+minus
+minus
minus
+minus
+wt site 1 mut site 1
lowast
Rela
tive l
ucife
rase
activ
ity
(b)
0020406081012
miR-NCmiR-136
+minus
+minus
minus
+minus
+wt site 2 mut site 2
lowastRe
lativ
e luc
ifera
se ac
tivity
(c)
0020406081012
miR-NCmiR-136
+minus
+minus
minus
+minus
+wt site 3 mut site 3
NS
Relat
ive l
ucife
rase
activ
ity
(d)
PPP2R2A
GAPDH
miR-136miR-NC mimics
(e)
Figure 3 PPP2R2A was a direct target of miR-136 (a) There were three potential miR-136 binding sites in PPP2R2A 31015840-UTR based on theTargetScan database the conservation of the miR-136 binding seed regions among different species was shown in shading and mutationswere shown in italics Fragments containing wild-type (wt) or mutant (mut) miR-136 binding sites in human PPP2R2A 31015840-UTR were cloneddownstreamof the luciferase reporter gene separately ((b)ndash(d)) Luciferase reporter assay (119899 = 3 for each group) Cos-7 cellswere cotransfectedwith a 31015840-UTR reporter construct and the miR-136 mimics or miR-NC and the results showed that site 1 and site 2 were the direct targets ofmiR-136 Luciferase activityrenilla activity was applied as the baseline control for the experiments using the same reporter Data representmean plusmn SD lowast119875 lt 005 (e) Western blot analyses of PPP2R2A expression in HaCaT cells transfected with miR-136 mimics or miR-NCGAPDH was used as loading control
PPP2R2A
GAPDH
Control TGF-1205731
(a)
0005101520253035 lowast
Control TGF-1205731Relat
ive P
PP2R
2A ex
pres
sion
(b)
Control NC PPP2R2A00020406081012 lowast
siRNA
Relat
ive P
PP2R
2A ex
pres
sion
(c)
Control0
02040608101214
TGF-1205731 +
lowast
TGF-1205731+
NC
OD
val
ue (450
nm)
PPP2R2A siRNA
(d)
Control
0
200
400
600
100
300
500
0 40 80 120 160
S 3820
G0G1 4906G2M 1274
0 40 80 120 1600
200
400
600
TGF-1205731 + NC
S 1367
G0G17889G2M 743
0
200
400
600
0 40 80 120 160TGF-1205731 +
PPP2R2A siRNA
S 2156
G0G1 6693G2M 1150
(e)
Figure 4 PPP2R2A was involved in TGF-1205731-induced proliferation arrest in HaCaT cells (a) Western blot analysis of PPP2R2A expression inresponse to TGF-1205731 treatment GAPDHwas used as internal control (b)Three independent results ofWestern blot were quantified by ImageJ (c) PPP2R2A silencing was verified by quantitative real-time PCR (d) Determination of cell viability with Cell Counting Kit-8 (e) Flowcytometry analysis of the cell cycle Bars indicate SD lowast119875 lt 005
BioMed Research International 7
TGF-1205731 PPP2R2AmiR-136
Keratinocytes proliferation
Figure 5 Schematic representation summarizing the roles of miR-136 and PPP2R2A during TGF-1205731-induced proliferation arrest Seetext for details
Recently results of others indicated that miR-136 enhancedphosphorylation of Erk12 through inhibition of PPP2R2Aexpression to promoted cell proliferation in human non-small cell lung cancer and the sequence at position 149ndash155 ofthe PPP2R2A 31015840-UTR was determined to be the target site ofmiR-136 [25] In this study we clarified miR-136 suppressedPPP2R2A expression by directly targeting two conservedregions at positions 149ndash155 and 712ndash719 in its 31015840-UTRFurthermore the protein level of PPP2R2A was significantlyreduced by miR-136 mimics transfection in HaCaT cellsTherefore we proposed that in the presence of TGF-1205731 miR-136 was a positive modulator of keratinocytes proliferationand PPP2R2A was a functional target of miR-136 via bindingto its 31015840-UTR region
In conclusion our results indicated that the miR-136treatment bypassed TGF-1205731-induced proliferation arrest atleast partially through downregulation of PPP2R2A expres-sion (Figure 5) Our data suggested that modulation ofkeratinocytes proliferation by miR-136 depends on the cel-lular environment especially in the presence of TGF-1205731which likely has major effects on the expression of miR-136target gene PPP2R2A Further studies are needed to deter-mine whether miR-136 based therapeutic strategies could beexploited to protect keratinocytes proliferation and functionin the condition of enhanced TGF-1205731 during wound healing
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Dianbao Zhang and Jing Wang contributed equally to thiswork
Acknowledgments
This work was funded by the National Basic ResearchProgram of China (2012CB518103) National Natural ScienceFoundation of China (81370883) Doctoral Fund of Ministryof Education of China (20132104110020) Science and Tech-nology Planning Project of Liaoning Province (2012225080)and Science and Technology Planning Project of Shenyang(F11-262-9-01) The authors thank Dr Hongchuan Xin andDr Ziwei Miao for critical reading and editing of this paper
References
[1] I Haase R Evans R Pofahl and F M Watt ldquoRegulation ofkeratinocyte shape migration and wound epithelialization byIGF-1 and EGF-dependent signalling pathwaysrdquo Journal of CellScience vol 116 no 15 pp 3227ndash3238 2003
[2] E Fuchs ldquoSkin stem cells rising to the surfacerdquo Journal of CellBiology vol 180 no 2 pp 273ndash284 2008
[3] C Pincelli andAMarconi ldquoKeratinocyte stem cells friends andfoesrdquo Journal of Cellular Physiology vol 225 no 2 pp 310ndash3152010
[4] J Nie X Fu and W Han ldquoMicroenvironment-dependenthomeostasis and differentiation of epidermal basal undiffer-entiated keratinocytes and their clinical applications in skinrepairrdquo Journal of the European Academy of Dermatology andVenereology vol 27 no 5 pp 531ndash535 2013
[5] S Guo and L A Dipietro ldquoFactors affecting wound healingrdquoJournal of Dental Research vol 89 no 3 pp 219ndash229 2010
[6] G S Schultz and AWysocki ldquoInteractions between extracellu-lar matrix and growth factors in wound healingrdquoWound Repairand Regeneration vol 17 no 2 pp 153ndash162 2009
[7] J S Kang C Liu andRDerynck ldquoNew regulatorymechanismsof TGF-120573 receptor functionrdquo Trends in Cell Biology vol 19 no8 pp 385ndash394 2009
[8] R Yi and E Fuchs ldquoMicroRNA-mediated control in the skinrdquoCell Death amp Differentiation vol 17 no 2 pp 229ndash235 2010
[9] M Teta Y S Choi T Okegbe et al ldquoInducible deletion of epi-dermal Dicer and Drosha reveals multiple functions for miR-NAs in postnatal skinrdquo Development vol 139 no 8 pp 1405ndash1416 2012
[10] D AntoniniM T Russo L de RosaMGorrese L del Vecchioand C Missero ldquoTranscriptional repression of miR-34 familycontributes to p63-mediated cell cycle progression in epidermalcellsrdquo Journal of Investigative Dermatology vol 130 no 5 pp1249ndash1257 2010
[11] N Xu P Brodin T Wei et al ldquoMiR-125b a MicroRNA down-regulated in psoriasis modulates keratinocyte proliferation bytargeting FGFR2rdquo Journal of Investigative Dermatology vol 131no 7 pp 1521ndash1529 2011
[12] Z Lin X Wang C Fewell J Cameron Q Yin and E KFlemington ldquoDifferential expression of the miR-200 familymicroRNAs in epithelial and B cells and regulation of Epstein-Barr virus reactivation by the miR-200 family member miR-429rdquo Journal of Virology vol 84 no 15 pp 7892ndash7897 2010
[13] A M Lena R Shalom-Feuerstein P R di Val Cervo et alldquomiR-203 represses ldquostemnessrdquo by repressingΔNp63rdquoCellDeathamp Differentiation vol 15 no 7 pp 1187ndash1195 2008
[14] S Biswas S Roy J Banerjee et al ldquoHypoxia inducible microR-NA 210 attenuates keratinocyte proliferation and impairs clo-sure in a murine model of ischemic woundsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 107 no 15 pp 6976ndash6981 2010
[15] M H Schmitz M Held V Janssens et al ldquoLive-cell imagingRNAi screen identifies PP2A-B55120572 and importin-120573 21 as keymitotic exit regulators in human cellsrdquo Nature Cell Biology vol12 no 9 pp 886ndash893 2010
[16] J Batut B Schmierer J Cao L A Raftery C S Hill and MHowell ldquoTwo highly related regulatory subunits of PP2A exertopposite effects on TGF-120573ActivinNodal signallingrdquo Develop-ment vol 135 no 17 pp 2927ndash2937 2008
[17] A Kurimchak and X Grana ldquoPP2A counterbalances phos-phorylation of pRB and mitotic proteins by multiple CDKs
8 BioMed Research International
potential implications for PP2A disruption in cancerrdquo Genes ampCancer vol 3 no 11-12 pp 739ndash748 2012
[18] G Jayadeva A Kurimchak J Garriga et al ldquoB55120572 PP2A hol-oenzymes modulate the phosphorylation status of the retino-blastomarelated protein p107 and its activationrdquo The Journal ofBiological Chemistry vol 285 no 39 pp 29863ndash29873 2010
[19] J M Overstreet R Samarakoon K K Meldrum and P JHiggins ldquoRedox control of p53 in the transcriptional regulationof TGF-1205731 target genes through SMAD cooperativityrdquo CellularSignalling vol 26 no 7 pp 1427ndash1436 2014
[20] M Motizuki K Isogaya K Miyake et al ldquoOligodendrocytetranscription factor 1 (Olig1) is a smad cofactor involved in cellmotility induced by transforming growth factor-120573rdquoThe Journalof Biological Chemistry vol 288 no 26 pp 18911ndash18922 2013
[21] K Rasanen and A Vaheri ldquoTGF-beta1 causes epithelial-mesenchymal transition in HaCaT derivatives but inducesexpression of COX-2 and migration only in benign not inmalignant keratinocytesrdquo Journal of Dermatological Science vol58 no 2 pp 97ndash104 2010
[22] G Viticchie A M Lena F Cianfarani et al ldquoMicroRNA-203contributes to skin re-epithelializationrdquo Cell Death amp Diseasevol 3 no 11 article e435 2012
[23] Y Yang J Wu H Guan et al ldquoMiR-136 promotes apoptosis ofglioma cells by targeting AEG-1 and Bcl-2rdquo FEBS Letters vol586 no 20 pp 3608ndash3612 2012
[24] D Y Lee Z Jeyapalan L Fang et al ldquoExpression of versican31015840-untranslated region modulates endogenous microrna func-tionsrdquo PLoS ONE vol 5 no 10 Article ID e13599 2010
[25] S Shen H Yue Y Li et al ldquoUpregulation of miR-136 in humannon-small cell lung cancer cells promotes Erk12 activation bytargeting PPP2R2Ardquo Tumor Biology vol 35 no 1 pp 631ndash6402014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
6 BioMed Research International
hsa-miR-136
wt site 2mut site 2PtrMmuRnoOcu
hsa-miR-136
wt site 1mut site 1PtrMmuRnoOcu
ACAUUGUUAUAGCUACAUGGAGA
ACAUUGUUAUAGCUACGGAACUA
ACAUUGUUAUAGCUACAUGGAGA
CACAUUUGAUAGCCUCAUGGAGA
CACAUUUGAUAGCCACAUGGAGA
AUUUUUAUAUAGCUACAUGGAGA
1471ndash1478Site 3
712ndash719Site 2
149ndash155Site 1CDS
PPP2R2A
5998400
3998400
5998400
5998400
5998400
59984005998400
5998400
3998400
3998400
3998400
3998400
3998400
3998400
| ||| | |
AGGUAGUAGUUUUGUUUACCUCA
AAAAGUGACAAAG--AAUGGAGA
AAAAGUGACAAAG--CGGAACUA
AAAAGUGACAAAG--AAUGGAGA
AAAAGUGACAAAG--AAUGGAGA
AAAAGUGACAAAG--AAUGGAGA
AAAAGUGACAAAG--AAUGGAGA
5998400
998400 998400
5998400
5998400
5998400
59984005998400
5
3998400
3998400
3998400
3998400
3998400
3998400
| ||||| | | | | |
3 AGGUAGUAGUUUUGUUUACCUCA hsa-miR-136
wt site 3mut site 3PtrMmuRnoOcu
UCUUGCAGAAACUUUAAUGGAGA
UCUUGCAGAAACUUUCGGAACUA
UCUUGCAGAAACUUUAAUGGAGA
GUCCUGCGGAAGCUUUAUGGAGA
GUCCUGCGAAAGCUUUAUGGAGA
GUCUUGAAGAGCUUUAAUGGAGA
5998400
998400 998400
5998400
5998400
5998400
59984005998400
5
3998400399840039984003998400
39984003998400
||| || ||
3 AGGUAGUAGUUUUGUUUACCUCA
(a)
0020406081012
miR-NCmiR-136
+minus
+minus
minus
+minus
+wt site 1 mut site 1
lowast
Rela
tive l
ucife
rase
activ
ity
(b)
0020406081012
miR-NCmiR-136
+minus
+minus
minus
+minus
+wt site 2 mut site 2
lowastRe
lativ
e luc
ifera
se ac
tivity
(c)
0020406081012
miR-NCmiR-136
+minus
+minus
minus
+minus
+wt site 3 mut site 3
NS
Relat
ive l
ucife
rase
activ
ity
(d)
PPP2R2A
GAPDH
miR-136miR-NC mimics
(e)
Figure 3 PPP2R2A was a direct target of miR-136 (a) There were three potential miR-136 binding sites in PPP2R2A 31015840-UTR based on theTargetScan database the conservation of the miR-136 binding seed regions among different species was shown in shading and mutationswere shown in italics Fragments containing wild-type (wt) or mutant (mut) miR-136 binding sites in human PPP2R2A 31015840-UTR were cloneddownstreamof the luciferase reporter gene separately ((b)ndash(d)) Luciferase reporter assay (119899 = 3 for each group) Cos-7 cellswere cotransfectedwith a 31015840-UTR reporter construct and the miR-136 mimics or miR-NC and the results showed that site 1 and site 2 were the direct targets ofmiR-136 Luciferase activityrenilla activity was applied as the baseline control for the experiments using the same reporter Data representmean plusmn SD lowast119875 lt 005 (e) Western blot analyses of PPP2R2A expression in HaCaT cells transfected with miR-136 mimics or miR-NCGAPDH was used as loading control
PPP2R2A
GAPDH
Control TGF-1205731
(a)
0005101520253035 lowast
Control TGF-1205731Relat
ive P
PP2R
2A ex
pres
sion
(b)
Control NC PPP2R2A00020406081012 lowast
siRNA
Relat
ive P
PP2R
2A ex
pres
sion
(c)
Control0
02040608101214
TGF-1205731 +
lowast
TGF-1205731+
NC
OD
val
ue (450
nm)
PPP2R2A siRNA
(d)
Control
0
200
400
600
100
300
500
0 40 80 120 160
S 3820
G0G1 4906G2M 1274
0 40 80 120 1600
200
400
600
TGF-1205731 + NC
S 1367
G0G17889G2M 743
0
200
400
600
0 40 80 120 160TGF-1205731 +
PPP2R2A siRNA
S 2156
G0G1 6693G2M 1150
(e)
Figure 4 PPP2R2A was involved in TGF-1205731-induced proliferation arrest in HaCaT cells (a) Western blot analysis of PPP2R2A expression inresponse to TGF-1205731 treatment GAPDHwas used as internal control (b)Three independent results ofWestern blot were quantified by ImageJ (c) PPP2R2A silencing was verified by quantitative real-time PCR (d) Determination of cell viability with Cell Counting Kit-8 (e) Flowcytometry analysis of the cell cycle Bars indicate SD lowast119875 lt 005
BioMed Research International 7
TGF-1205731 PPP2R2AmiR-136
Keratinocytes proliferation
Figure 5 Schematic representation summarizing the roles of miR-136 and PPP2R2A during TGF-1205731-induced proliferation arrest Seetext for details
Recently results of others indicated that miR-136 enhancedphosphorylation of Erk12 through inhibition of PPP2R2Aexpression to promoted cell proliferation in human non-small cell lung cancer and the sequence at position 149ndash155 ofthe PPP2R2A 31015840-UTR was determined to be the target site ofmiR-136 [25] In this study we clarified miR-136 suppressedPPP2R2A expression by directly targeting two conservedregions at positions 149ndash155 and 712ndash719 in its 31015840-UTRFurthermore the protein level of PPP2R2A was significantlyreduced by miR-136 mimics transfection in HaCaT cellsTherefore we proposed that in the presence of TGF-1205731 miR-136 was a positive modulator of keratinocytes proliferationand PPP2R2A was a functional target of miR-136 via bindingto its 31015840-UTR region
In conclusion our results indicated that the miR-136treatment bypassed TGF-1205731-induced proliferation arrest atleast partially through downregulation of PPP2R2A expres-sion (Figure 5) Our data suggested that modulation ofkeratinocytes proliferation by miR-136 depends on the cel-lular environment especially in the presence of TGF-1205731which likely has major effects on the expression of miR-136target gene PPP2R2A Further studies are needed to deter-mine whether miR-136 based therapeutic strategies could beexploited to protect keratinocytes proliferation and functionin the condition of enhanced TGF-1205731 during wound healing
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Dianbao Zhang and Jing Wang contributed equally to thiswork
Acknowledgments
This work was funded by the National Basic ResearchProgram of China (2012CB518103) National Natural ScienceFoundation of China (81370883) Doctoral Fund of Ministryof Education of China (20132104110020) Science and Tech-nology Planning Project of Liaoning Province (2012225080)and Science and Technology Planning Project of Shenyang(F11-262-9-01) The authors thank Dr Hongchuan Xin andDr Ziwei Miao for critical reading and editing of this paper
References
[1] I Haase R Evans R Pofahl and F M Watt ldquoRegulation ofkeratinocyte shape migration and wound epithelialization byIGF-1 and EGF-dependent signalling pathwaysrdquo Journal of CellScience vol 116 no 15 pp 3227ndash3238 2003
[2] E Fuchs ldquoSkin stem cells rising to the surfacerdquo Journal of CellBiology vol 180 no 2 pp 273ndash284 2008
[3] C Pincelli andAMarconi ldquoKeratinocyte stem cells friends andfoesrdquo Journal of Cellular Physiology vol 225 no 2 pp 310ndash3152010
[4] J Nie X Fu and W Han ldquoMicroenvironment-dependenthomeostasis and differentiation of epidermal basal undiffer-entiated keratinocytes and their clinical applications in skinrepairrdquo Journal of the European Academy of Dermatology andVenereology vol 27 no 5 pp 531ndash535 2013
[5] S Guo and L A Dipietro ldquoFactors affecting wound healingrdquoJournal of Dental Research vol 89 no 3 pp 219ndash229 2010
[6] G S Schultz and AWysocki ldquoInteractions between extracellu-lar matrix and growth factors in wound healingrdquoWound Repairand Regeneration vol 17 no 2 pp 153ndash162 2009
[7] J S Kang C Liu andRDerynck ldquoNew regulatorymechanismsof TGF-120573 receptor functionrdquo Trends in Cell Biology vol 19 no8 pp 385ndash394 2009
[8] R Yi and E Fuchs ldquoMicroRNA-mediated control in the skinrdquoCell Death amp Differentiation vol 17 no 2 pp 229ndash235 2010
[9] M Teta Y S Choi T Okegbe et al ldquoInducible deletion of epi-dermal Dicer and Drosha reveals multiple functions for miR-NAs in postnatal skinrdquo Development vol 139 no 8 pp 1405ndash1416 2012
[10] D AntoniniM T Russo L de RosaMGorrese L del Vecchioand C Missero ldquoTranscriptional repression of miR-34 familycontributes to p63-mediated cell cycle progression in epidermalcellsrdquo Journal of Investigative Dermatology vol 130 no 5 pp1249ndash1257 2010
[11] N Xu P Brodin T Wei et al ldquoMiR-125b a MicroRNA down-regulated in psoriasis modulates keratinocyte proliferation bytargeting FGFR2rdquo Journal of Investigative Dermatology vol 131no 7 pp 1521ndash1529 2011
[12] Z Lin X Wang C Fewell J Cameron Q Yin and E KFlemington ldquoDifferential expression of the miR-200 familymicroRNAs in epithelial and B cells and regulation of Epstein-Barr virus reactivation by the miR-200 family member miR-429rdquo Journal of Virology vol 84 no 15 pp 7892ndash7897 2010
[13] A M Lena R Shalom-Feuerstein P R di Val Cervo et alldquomiR-203 represses ldquostemnessrdquo by repressingΔNp63rdquoCellDeathamp Differentiation vol 15 no 7 pp 1187ndash1195 2008
[14] S Biswas S Roy J Banerjee et al ldquoHypoxia inducible microR-NA 210 attenuates keratinocyte proliferation and impairs clo-sure in a murine model of ischemic woundsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 107 no 15 pp 6976ndash6981 2010
[15] M H Schmitz M Held V Janssens et al ldquoLive-cell imagingRNAi screen identifies PP2A-B55120572 and importin-120573 21 as keymitotic exit regulators in human cellsrdquo Nature Cell Biology vol12 no 9 pp 886ndash893 2010
[16] J Batut B Schmierer J Cao L A Raftery C S Hill and MHowell ldquoTwo highly related regulatory subunits of PP2A exertopposite effects on TGF-120573ActivinNodal signallingrdquo Develop-ment vol 135 no 17 pp 2927ndash2937 2008
[17] A Kurimchak and X Grana ldquoPP2A counterbalances phos-phorylation of pRB and mitotic proteins by multiple CDKs
8 BioMed Research International
potential implications for PP2A disruption in cancerrdquo Genes ampCancer vol 3 no 11-12 pp 739ndash748 2012
[18] G Jayadeva A Kurimchak J Garriga et al ldquoB55120572 PP2A hol-oenzymes modulate the phosphorylation status of the retino-blastomarelated protein p107 and its activationrdquo The Journal ofBiological Chemistry vol 285 no 39 pp 29863ndash29873 2010
[19] J M Overstreet R Samarakoon K K Meldrum and P JHiggins ldquoRedox control of p53 in the transcriptional regulationof TGF-1205731 target genes through SMAD cooperativityrdquo CellularSignalling vol 26 no 7 pp 1427ndash1436 2014
[20] M Motizuki K Isogaya K Miyake et al ldquoOligodendrocytetranscription factor 1 (Olig1) is a smad cofactor involved in cellmotility induced by transforming growth factor-120573rdquoThe Journalof Biological Chemistry vol 288 no 26 pp 18911ndash18922 2013
[21] K Rasanen and A Vaheri ldquoTGF-beta1 causes epithelial-mesenchymal transition in HaCaT derivatives but inducesexpression of COX-2 and migration only in benign not inmalignant keratinocytesrdquo Journal of Dermatological Science vol58 no 2 pp 97ndash104 2010
[22] G Viticchie A M Lena F Cianfarani et al ldquoMicroRNA-203contributes to skin re-epithelializationrdquo Cell Death amp Diseasevol 3 no 11 article e435 2012
[23] Y Yang J Wu H Guan et al ldquoMiR-136 promotes apoptosis ofglioma cells by targeting AEG-1 and Bcl-2rdquo FEBS Letters vol586 no 20 pp 3608ndash3612 2012
[24] D Y Lee Z Jeyapalan L Fang et al ldquoExpression of versican31015840-untranslated region modulates endogenous microrna func-tionsrdquo PLoS ONE vol 5 no 10 Article ID e13599 2010
[25] S Shen H Yue Y Li et al ldquoUpregulation of miR-136 in humannon-small cell lung cancer cells promotes Erk12 activation bytargeting PPP2R2Ardquo Tumor Biology vol 35 no 1 pp 631ndash6402014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
BioMed Research International 7
TGF-1205731 PPP2R2AmiR-136
Keratinocytes proliferation
Figure 5 Schematic representation summarizing the roles of miR-136 and PPP2R2A during TGF-1205731-induced proliferation arrest Seetext for details
Recently results of others indicated that miR-136 enhancedphosphorylation of Erk12 through inhibition of PPP2R2Aexpression to promoted cell proliferation in human non-small cell lung cancer and the sequence at position 149ndash155 ofthe PPP2R2A 31015840-UTR was determined to be the target site ofmiR-136 [25] In this study we clarified miR-136 suppressedPPP2R2A expression by directly targeting two conservedregions at positions 149ndash155 and 712ndash719 in its 31015840-UTRFurthermore the protein level of PPP2R2A was significantlyreduced by miR-136 mimics transfection in HaCaT cellsTherefore we proposed that in the presence of TGF-1205731 miR-136 was a positive modulator of keratinocytes proliferationand PPP2R2A was a functional target of miR-136 via bindingto its 31015840-UTR region
In conclusion our results indicated that the miR-136treatment bypassed TGF-1205731-induced proliferation arrest atleast partially through downregulation of PPP2R2A expres-sion (Figure 5) Our data suggested that modulation ofkeratinocytes proliferation by miR-136 depends on the cel-lular environment especially in the presence of TGF-1205731which likely has major effects on the expression of miR-136target gene PPP2R2A Further studies are needed to deter-mine whether miR-136 based therapeutic strategies could beexploited to protect keratinocytes proliferation and functionin the condition of enhanced TGF-1205731 during wound healing
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Dianbao Zhang and Jing Wang contributed equally to thiswork
Acknowledgments
This work was funded by the National Basic ResearchProgram of China (2012CB518103) National Natural ScienceFoundation of China (81370883) Doctoral Fund of Ministryof Education of China (20132104110020) Science and Tech-nology Planning Project of Liaoning Province (2012225080)and Science and Technology Planning Project of Shenyang(F11-262-9-01) The authors thank Dr Hongchuan Xin andDr Ziwei Miao for critical reading and editing of this paper
References
[1] I Haase R Evans R Pofahl and F M Watt ldquoRegulation ofkeratinocyte shape migration and wound epithelialization byIGF-1 and EGF-dependent signalling pathwaysrdquo Journal of CellScience vol 116 no 15 pp 3227ndash3238 2003
[2] E Fuchs ldquoSkin stem cells rising to the surfacerdquo Journal of CellBiology vol 180 no 2 pp 273ndash284 2008
[3] C Pincelli andAMarconi ldquoKeratinocyte stem cells friends andfoesrdquo Journal of Cellular Physiology vol 225 no 2 pp 310ndash3152010
[4] J Nie X Fu and W Han ldquoMicroenvironment-dependenthomeostasis and differentiation of epidermal basal undiffer-entiated keratinocytes and their clinical applications in skinrepairrdquo Journal of the European Academy of Dermatology andVenereology vol 27 no 5 pp 531ndash535 2013
[5] S Guo and L A Dipietro ldquoFactors affecting wound healingrdquoJournal of Dental Research vol 89 no 3 pp 219ndash229 2010
[6] G S Schultz and AWysocki ldquoInteractions between extracellu-lar matrix and growth factors in wound healingrdquoWound Repairand Regeneration vol 17 no 2 pp 153ndash162 2009
[7] J S Kang C Liu andRDerynck ldquoNew regulatorymechanismsof TGF-120573 receptor functionrdquo Trends in Cell Biology vol 19 no8 pp 385ndash394 2009
[8] R Yi and E Fuchs ldquoMicroRNA-mediated control in the skinrdquoCell Death amp Differentiation vol 17 no 2 pp 229ndash235 2010
[9] M Teta Y S Choi T Okegbe et al ldquoInducible deletion of epi-dermal Dicer and Drosha reveals multiple functions for miR-NAs in postnatal skinrdquo Development vol 139 no 8 pp 1405ndash1416 2012
[10] D AntoniniM T Russo L de RosaMGorrese L del Vecchioand C Missero ldquoTranscriptional repression of miR-34 familycontributes to p63-mediated cell cycle progression in epidermalcellsrdquo Journal of Investigative Dermatology vol 130 no 5 pp1249ndash1257 2010
[11] N Xu P Brodin T Wei et al ldquoMiR-125b a MicroRNA down-regulated in psoriasis modulates keratinocyte proliferation bytargeting FGFR2rdquo Journal of Investigative Dermatology vol 131no 7 pp 1521ndash1529 2011
[12] Z Lin X Wang C Fewell J Cameron Q Yin and E KFlemington ldquoDifferential expression of the miR-200 familymicroRNAs in epithelial and B cells and regulation of Epstein-Barr virus reactivation by the miR-200 family member miR-429rdquo Journal of Virology vol 84 no 15 pp 7892ndash7897 2010
[13] A M Lena R Shalom-Feuerstein P R di Val Cervo et alldquomiR-203 represses ldquostemnessrdquo by repressingΔNp63rdquoCellDeathamp Differentiation vol 15 no 7 pp 1187ndash1195 2008
[14] S Biswas S Roy J Banerjee et al ldquoHypoxia inducible microR-NA 210 attenuates keratinocyte proliferation and impairs clo-sure in a murine model of ischemic woundsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 107 no 15 pp 6976ndash6981 2010
[15] M H Schmitz M Held V Janssens et al ldquoLive-cell imagingRNAi screen identifies PP2A-B55120572 and importin-120573 21 as keymitotic exit regulators in human cellsrdquo Nature Cell Biology vol12 no 9 pp 886ndash893 2010
[16] J Batut B Schmierer J Cao L A Raftery C S Hill and MHowell ldquoTwo highly related regulatory subunits of PP2A exertopposite effects on TGF-120573ActivinNodal signallingrdquo Develop-ment vol 135 no 17 pp 2927ndash2937 2008
[17] A Kurimchak and X Grana ldquoPP2A counterbalances phos-phorylation of pRB and mitotic proteins by multiple CDKs
8 BioMed Research International
potential implications for PP2A disruption in cancerrdquo Genes ampCancer vol 3 no 11-12 pp 739ndash748 2012
[18] G Jayadeva A Kurimchak J Garriga et al ldquoB55120572 PP2A hol-oenzymes modulate the phosphorylation status of the retino-blastomarelated protein p107 and its activationrdquo The Journal ofBiological Chemistry vol 285 no 39 pp 29863ndash29873 2010
[19] J M Overstreet R Samarakoon K K Meldrum and P JHiggins ldquoRedox control of p53 in the transcriptional regulationof TGF-1205731 target genes through SMAD cooperativityrdquo CellularSignalling vol 26 no 7 pp 1427ndash1436 2014
[20] M Motizuki K Isogaya K Miyake et al ldquoOligodendrocytetranscription factor 1 (Olig1) is a smad cofactor involved in cellmotility induced by transforming growth factor-120573rdquoThe Journalof Biological Chemistry vol 288 no 26 pp 18911ndash18922 2013
[21] K Rasanen and A Vaheri ldquoTGF-beta1 causes epithelial-mesenchymal transition in HaCaT derivatives but inducesexpression of COX-2 and migration only in benign not inmalignant keratinocytesrdquo Journal of Dermatological Science vol58 no 2 pp 97ndash104 2010
[22] G Viticchie A M Lena F Cianfarani et al ldquoMicroRNA-203contributes to skin re-epithelializationrdquo Cell Death amp Diseasevol 3 no 11 article e435 2012
[23] Y Yang J Wu H Guan et al ldquoMiR-136 promotes apoptosis ofglioma cells by targeting AEG-1 and Bcl-2rdquo FEBS Letters vol586 no 20 pp 3608ndash3612 2012
[24] D Y Lee Z Jeyapalan L Fang et al ldquoExpression of versican31015840-untranslated region modulates endogenous microrna func-tionsrdquo PLoS ONE vol 5 no 10 Article ID e13599 2010
[25] S Shen H Yue Y Li et al ldquoUpregulation of miR-136 in humannon-small cell lung cancer cells promotes Erk12 activation bytargeting PPP2R2Ardquo Tumor Biology vol 35 no 1 pp 631ndash6402014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
8 BioMed Research International
potential implications for PP2A disruption in cancerrdquo Genes ampCancer vol 3 no 11-12 pp 739ndash748 2012
[18] G Jayadeva A Kurimchak J Garriga et al ldquoB55120572 PP2A hol-oenzymes modulate the phosphorylation status of the retino-blastomarelated protein p107 and its activationrdquo The Journal ofBiological Chemistry vol 285 no 39 pp 29863ndash29873 2010
[19] J M Overstreet R Samarakoon K K Meldrum and P JHiggins ldquoRedox control of p53 in the transcriptional regulationof TGF-1205731 target genes through SMAD cooperativityrdquo CellularSignalling vol 26 no 7 pp 1427ndash1436 2014
[20] M Motizuki K Isogaya K Miyake et al ldquoOligodendrocytetranscription factor 1 (Olig1) is a smad cofactor involved in cellmotility induced by transforming growth factor-120573rdquoThe Journalof Biological Chemistry vol 288 no 26 pp 18911ndash18922 2013
[21] K Rasanen and A Vaheri ldquoTGF-beta1 causes epithelial-mesenchymal transition in HaCaT derivatives but inducesexpression of COX-2 and migration only in benign not inmalignant keratinocytesrdquo Journal of Dermatological Science vol58 no 2 pp 97ndash104 2010
[22] G Viticchie A M Lena F Cianfarani et al ldquoMicroRNA-203contributes to skin re-epithelializationrdquo Cell Death amp Diseasevol 3 no 11 article e435 2012
[23] Y Yang J Wu H Guan et al ldquoMiR-136 promotes apoptosis ofglioma cells by targeting AEG-1 and Bcl-2rdquo FEBS Letters vol586 no 20 pp 3608ndash3612 2012
[24] D Y Lee Z Jeyapalan L Fang et al ldquoExpression of versican31015840-untranslated region modulates endogenous microrna func-tionsrdquo PLoS ONE vol 5 no 10 Article ID e13599 2010
[25] S Shen H Yue Y Li et al ldquoUpregulation of miR-136 in humannon-small cell lung cancer cells promotes Erk12 activation bytargeting PPP2R2Ardquo Tumor Biology vol 35 no 1 pp 631ndash6402014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom