role of host mirna hsa-mir-139-3p in hpv-16 induced …biology of human tumors role of host mirna...

Biology of Human Tumors

Role of Host miRNA Hsa-miR-139-3p inHPV-16–Induced CarcinomasM.K. Sannigrahi1, Rajni Sharma2, Varinder Singh1, Naresh K. Panda1,Vidya Rattan3, and Madhu Khullar2

Abstract

Purpose:Human papillomavirus 16 (HPV-16) is an importantrisk factor in head and neck cancer (HNC). Studies suggest thatmiRNAs play an important role in cancer; however, their role inHPV-mediated oncogenesis remains largely unknown. We inves-tigated the role ofmiRNAswithHPV-16 as putative target inHPV-16–mediated cancers.

Experimental Design: Using in silico tools, we identifiedmiRNAs with putative binding sequences on HPV-16 miRNAs.Hsa-miR-139-3p was identified as best candidate miRNA byluciferase reporter assay and was found to be significantlydownregulated in HPV-16–positive tissues and cell lines. Over-expression/inhibition studies were performed to determine therole of miRNA in regulating oncogenic pathways.

Results:Hsa-miR-139-3pwas found to target high-riskHPV-16oncogenic proteins and revive major tumor suppressor proteins(p53, p21, and p16). This resulted in inhibition of cell prolifer-

ation and cell migration, cell-cycle arrest at G2–M phase andincreased cell death of HPV-16–positive cells. Analysis of TheCancer Genome Atlas (TCGA) data showed decreased expressionof Hsa-miR-139-3p in HPV-16–positive HNC and cervical cancercases, and its higher expression correlated with better survivaloutcome in both cases. Increased DNA methylation of Hsa-miR-139-3p harboring gene PDE2A at its promoter/CpG islandswas observed in HPV-16–positive tissues and cell lines, whichfurther correlated with Hsa-miR-139-3p expression, suggestingits role in regulating Hsa-miR-139-3p expression. Furthermore,we observed an increased sensitization of Hsa-miR-139-3p over-expressed HPV-16–positive cells to chemotherapeutic drugs (cis-platin and 5-fluorouracil).

Conclusions: HPV-16–mediated downregulation of Hsa-miR-139-3p may promote oncogenesis in HNC and cervicalcancer. Clin Cancer Res; 23(14); 3884–95. �2017 AACR.

IntroductionHuman papillomaviruses (HPV) represents an important eti-

ological agent for cervical cancer and head and neck cancer. Thevirus expresses twomainoncogenic proteins, E6 andE7 that targethost tumor suppressor proteins p53 and pRB, respectively (1, 2).Besides this, HPVs also induce various genetic and epigeneticchanges in host leading to carcinogenesis (3).

Virus–host interactions are critical in determining host suscep-tibility/resistance to viral infections (4, 5). miRNA form animportant part of mammalian innate antiviral immunityresponse and were found to act as restriction factors to limit

infection by several types of viruses like human immunodeficien-cy virus (6), primate foamy virus type 1 (7), and vesicularstomatitis virus (8). It is suggested that viruses may altermiRNA expression of infected cells, resulting in deregulation ofhost defense pathways and helping invading viruses to establishan environment favorable for persistence of viral infection (9, 10).

Vertebrates encode several hundred miRNA families withdistinct seed sequences and it has been suggested that two-thirdof these vertebrate miRNAs may have at least one hit in a virusgenome of approximately 16 kb by random chance alone (11).Furthermore, knocking down the miRNA-processing enzymessuch as Drosha and Dicer which reduces the processing ofmature mammalian miRNAs has been shown to lead to morerobust viral replication indicating their role in host–viral inter-actions (12–14). Furthermore, infecting viruses have beenshown to alter miRNA expression of infected cells, resulting inde-regulation of host defense pathways and helping invadingviruses to establish an environment favorable for the persistenceof viral infection (15). Several recent studies have reporteddifferential expression of microRNAs in HPV infected cell linesand HPV-positive tumor tissues (16–18) and it has been sug-gested that aberrant miRNA expression may be regulated byepigenetic mechanisms like DNA methylation in HPV-asso-ciated cancers (19). We hypothesized that HPV-16–induceddifferential expression of host miRNAs may help its survivaland activation of HPV-16 associated oncogenic pathways. Ourresults show that downregulation of Hsa-miR-139-3p, targetingHPV-16 viral transcription, activates HPV-16 oncogenic E6and E7 proteins, and may be involved in HPV-16–inducedcarcinogenesis.

1Department of Otolaryngology, Post Graduate Institute of Medical Educationand Research (PGIMER), Chandigarh, India. 2Department of Experimental Med-icine and Biotechnology, PGIMER, Chandigarh, India. 3Unit of Oral HealthSciences, PGIMER, Chandigarh, India.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Prior presentations: The work has been presented in various national andinternational conferences. Part of the work is published as an abstract inconference proceedings, including AACR Special Conference on NoncodingRNAs and Cancer: Mechanisms toMedicines; December 4–7, 2015, Boston, MA, aconference on new ideas in cancer-challenging dogmas, and 35th AnnualConvention of Indian Association for Cancer Research.

Corresponding Author: Madhu Khullar, Post Graduate Institute of MedicalEducation and Research, Chandigarh 160012, India. Phone: 172-274-2842; Fax:172-274-4401; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-16-2936

�2017 American Association for Cancer Research.

ClinicalCancerResearch

Clin Cancer Res; 23(14) July 15, 20173884

on February 22, 2021. © 2017 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst January 31, 2017; DOI: 10.1158/1078-0432.CCR-16-2936

http://clincancerres.aacrjournals.org/

Materials and MethodsPatient specimens and cell cultures

HNC tissue samples were collected from patients with sus-pected malignancy attending minor OT, Department of Otolar-yngology, PGIMER (Chandigarh, India), after obtaining informedconsent from individual patients. Control tissue group consistedof patients undergoing surgery for non-neoplastic diseases ofhead and neck in Department of Oral Health Sciences, PGIMER(Chandigarh, India).

Three HPV-negative cell-lines (UPCI:SCC-116, HaCat, andHEK-293) and three HPV-16–positive cell-lines (UPCI:SCC-090, SiHa, and CaSki) were cultured (20).

HPV-16 detection in tissue samplesHPV-16 was detected in DNA samples using: MY09-MY11 pri-

mers,Gp5þ/Gp6þprimers, andHPV-16-E6-specificprimers.HPV-16viral load and relative expressionofHPV-16-E7mRNAwasmeasuredby using qRT-PCR (ref. 21; Supplementary Information S1).

In silico identification of miRNAs targeting HPV-16The seed sequences of the miRNAs were screened for ones that

could potentially directly target HPV-16 mRNAs using MicroIn-spector (22), RegRNA program (23), and RNAHybrid program(24). PutativemiRNA targets on host mRNAswere predicted usingTargetScan software version 6.2 (25). Out of the >600 miRNAs

obtained from RegRNA program and Micro-Inspector programhaving putative target in HPV-16 mRNAs, those miRNAs(�100 miRNAs) having 2–8 seed sequence at the 30-end andwith binding free energies under the threshold of �20.0 kcal/mol were initially identified and screened for their HPV-16mRNA specificity by RNA-Hybrid program and for putativetargets on host mRNAs by TargetScan software version 6.2.10 best miRNAs were shortlisted on the basis of high numberof target sites in HPV-16 mRNAs and least putative target sites inhuman mRNAs (Supplementary Information S2). Primary cul-ture of keratinocytes were used as control (SupplementaryInformation S3) and differential expression of these miRNAswere further analyzed under different conditions (Supplemen-tary Information S4) to identify best candidate miRNAs havingputative target on HPV-16 mRNAs.

IHC staining, Western blot analysis, and antibodiesTissue samples were examined by IHC/Western blotting (WB)

for HPV-16-specific pathway proteins using Anti-p16 (sc-9968;Santa Cruz Biotechnology), anti-p53 (sc-6243; Santa Cruz Bio-technology), anti-pRb (sc-73598; Santa Cruz Biotechnology),anti-E6 (sc-460; Santa Cruz Biotechnology), anti-p21 (sc-6246;Santa Cruz Biotechnology), and anti-GAPDH (sc-25778; SantaCruz Biotechnology) antibodies. Quantification was done usingImageJ software (Supplementary Information S5).

Transfection with Hsa-miR-139-3pmimic/inhibitor/scrambledcontrol

Hsa-miR-139-3pmimic (referred asmimic, catalog no. 4464084;Thermo Fisher Scientific) and Hsa-miR-139-3p inhibitor (referredas inhibitor, catalog no. 4464066; Thermo Fisher Scientific) wereused to accordingly increase and decrease endogenous Hsa-miR-139-3p levels, respectably by transfection using Lipofectamine2000 (catalog no. 11668; Invitrogen). Cells were treated with100 pmol/mL of mimic/inhibitor. Scrambled miRNA (catalog no.4464058; Thermo Fisher Scientific) was used as negative control.

Dual luciferase assayLuciferase assaywas performed inHPV-negativeUPCI:SCC116

cells using noncommercial dual-luciferase enzyme assay system(26). Firefly luciferase (FL) plasmid pMiR-HPV16-E7 (with region562 to 658 encoding E7-CDS, Addgene #53696) and pMiR-HPV16-E1 (with region 865 to 2813 encoding E1-CDS, Addgene#53699; ref. 27), and Renilla pRL-SV40P (Addgene #27163;ref. 28). Renilla luciferase pRL-SV40P plasmid was used as aninternal control. Five-hundred nanograms of each plasmid and100 pmol of Hsa-miR-139-3p scrambled/mimic/inhibitor wastransfected into cells.

Cell proliferation, migration, and apoptosis analysisCell proliferation was measured using Click-iT Plus EdU cell

proliferation assay (catalog no. C10632; Life Technologies;ref. 29). Cell death and cell cycle was determined by Dead CellApoptosis Kit with Annexin V FITC and PI (catalog no. V13242;Life Technologies). Cells were examined in a FACSCalibur flowcytometer (BD Biosciences). Cell migration was analyzed byin vitro scratch assay (30). The width of scratch wounds was mea-sured using ImageJ software (Supplementary Information S6).

TCGA casesmiRNA profile and clinical details of HNC and CC patients

of TCGA were obtained from Firehose Broad GDAC (http://gdac.

Translational RelevanceHPV-16 is one of the emerging and major risk factors in

head and neck cancer and cervical cancer. We have shown anovel viral–host interaction mechanism through cross-talkbetween HPV-16 and Hsa-miR-139-3p. Putative miRNAs tar-geting HPV-16mRNAwere identified and validated in clinicalsamples, TCGA database, and in vitro studies in head and neckcancer and cervical cancer cases. We observed that* Hsa-miR-139-3p had four putative targets in E1 region of

HPV-16 early mRNA.* Hsa-miR-139-3p overexpression resulted in decreased

expression of HPV-16 mRNAs/proteins (E6/E7).* Hsa-miR-139-3p overexpression also resulted in decreased

p16, revival of p53 and p21 expression.* Hsa-miR-139-3p overexpression decreased cell

proliferation, cell migration, and G2–M arrest, leading tocell death.

* Decreased expression of Hsa-miR-139-3p is due topromoter methylation of its gene.

* Hsa-miR-139-3p sensitizes HPV-16–positive cells tochemotherapy.

We propose that HPV-16–mediated promoter methylationof Hsa-miR-139-3p gene may result in downregulation ofHsa-miR-139-3p in HPV-16–infected cells, leading to upregu-lation of pro-oncogenic pathways and HPV-16-induced car-cinogenesis. Thus, identification of such antiviral miRNAsthat are epigenetically silenced by HPV-16 will help in devel-oping epigenetic therapy againstHPV-induced cancer. Further,using such miRNAs in combination with common che-motherapies can lead to de-escalation of dose of chemo-drugsand will be beneficial to patients.

Host miRNAs Targeting HPV-16 HNSCC

www.aacrjournals.org Clin Cancer Res; 23(14) July 15, 2017 3885



http://gdac.broadinstitute.org/


broadinstitute.org/). A total of 255 cervical squamous cell carci-noma cases were included for analysis. A total of 279 HNCsamples already analyzed for HPV (31, 32) were included instudy, which were further grouped into HPV-negative HNC(n ¼ 243) and HPV-16–positive (n ¼ 29). Other HPV typepositive cases (n ¼ 7) were excluded. Methylation data of HNC(n ¼ 261) and CC (n ¼ 177) cases were obtained from MethHCdatabase (ref. 33; Supplementary Information S7).

DNA methylation and acetylation analysisCells were treated with 5-Aza-20-deoxycytidine (10 mmol/L)

and Trichostatin A (100 ng/mL) for 4 days, andmedia containing5-aza-dC/TSA was changed every 24 hours. For methylationanalysis, Genomic DNA (200 ng) converted with the EZ-DNAMethylation Kit (Zymo Research) and amplified using methyla-tion-specific Hsa-miR-139-3p primers and TaKaRa EpiTaq HS(catalog no. R100A; Clontech Laboratories). For acetylation anal-ysis, chromatin immunoprecipitation (ChIP) assays were per-formed using ChIP-IT Express Enzymatic Kit (catalog no.53009; Active-Motif; Supplementary Information S8).

In vitro chemosensitivity assayCells were treated with different doses of cisplatin (CDDP) and

5-fluorouracil (5-FU) combination for 48 hours and cell viabilitywas determined using MTT assay to calculate IC50/LD50. Finally,their effect was observed in combination with Hsa-miR-139-3p

overexpression/inhibition. In brief, cell lines with 4,000 cells/wellwere seeded in 96-well plates were transfected with 50 pmol Hsa-miR-139-3p mimic/inhibitor. After 24 hours, cells were treatedwith 5, 10, and 20mmol/L of chemotherapeutic drugs for 48hoursand cell viability wasmeasured (Supplementary Information S9).

Statistical analysisStatistical tests like nonparametric Mann–Whitney test, two-

tailed t test, ANOVA, and survival analysis done using GraphPadPrism 5 (Graph Pad Software). A P value of <0.05 was denoted astatistically significant difference.

Ethical approvalApproved by Institutional Ethics Committee (IEC), Post Grad-

uate Institute of Medical Education and Research (PGIMER),Chandigarh, India.

ResultsA total of 110 suspected cases of HNC were analyzed for HPV/

HPV-16 infection using PCR (Supplementary Information S1)and were grouped into HPV-negative HNC (n ¼ 40), Control(HPV-negative, n ¼ 40), and HPV-16–positive HNC (n ¼ 30).HPV-16DNA–positivepatientswere further grouped intoHPV-16E7 mRNA positive (n¼ 20) and HPV-16 E7 mRNA negative (n ¼10). The demographic profile of the patients is shown in Table 1.

Table 1. Demographic characteristics of patients

Groups Hsa-miR-139-3p expression HPV-16 E7 mRNA-positive group

Patient characteristics Control HPV-16–negative HNC HPV-16–positive HNCHsa-miR-139-3p upE7 mRNA-positive

Hsa-miR-139-3p downE7 mRNA-negative P value

Total HNC patients 40 40 30 20 10Age <60 y 32 (80) 33 (82.5) 25 (83.3) 17 (85) 8 (80) 0.551�60 y 8 (20) 7 (17.5) 5 (16.7) 3 (15) 2 (20)GenderMale 30 (75) 32 (80) 25 (83.3) 18 (90) 7 (70) 0.191Female 10 (25) 8 (20) 5 (16.7) 2 (10) 3 (30)

SubsiteOral cavity 15 (37.5) 12 (40) 7 (35) 5 (50)Oropharynx NIL 14 (35) 13 (43) 8 (40) 5 (50)Hypopharynx 5 (12.5) 2 (7) 2 (10) 0Larynx 5 (12.5) 3 (10) 3 (15) 0

Overall stageStage 0 þ 1 NIL 0 0 0 0 1Stage 2þ3þ4 40 (100) 30 (100) 20 (100) 10 (100)

SmokingNever smokers 10 (25) 5 (12.5) 5 (16.7) 4 (20) 1 (10) 1Light smokers 12 (30) 8 (20) 10 (33.3) 6 (30) 4 (40) 0.600Heavy smokers 18 (45) 27 (67.5) 15 (50) 10 (50) 5 (50) 1

AlcoholNo 20 (50) 15 (37.5) 17 (56.7) 10 (50) 7 (70) 0.440Yes 10 (25) 25 (62.5) 13 (43.3) 10 (50) 3 (30)

ChewingNon-chewers 32 (80) 28 (70) 22 (73.3) 15 (75) 7 (70) 1Chewers 8 (20) 12 (30) 8 (26.7) 5 (25) 3 (30)

Viral loadHigh NIL NIL 14 (46.7) 12 (60) 2 (20) 0.044a

Low 16 (53.3) 8 (40) 8 (80)IntegrationIntegrated NIL NIL 15 (50) 13 (65) 2 (20) 0.002a

Mixed 8 (26.7) 6 (30) 2 (20) 0.040a

Episomal 7 (23.3) 1 (5) 6 (60) 1p16 stainingPositive NIL NIL 14 (46.7) 14 (70) 0 0.000a

Negative 16 (54.3) 6 (30) 10 (100)aStatistically significant, P < 0.05.

Sannigrahi et al.

Clin Cancer Res; 23(14) July 15, 2017 Clinical Cancer Research3886



http://gdac.broadinstitute.org/


In silico selection of host miRNAs with HPV-16 mRNAs asputative targets

miRNAs with binding sites in HPV-16 mRNAs were screenedbased on their binding free energies (< �20.0 kcal/mol), "seedregion" (six to eight nucleotides at 50 end) and Hydrogen bondindex. They were shortlisted on the basis of high number of targetsites in HPV-16 mRNAs and least putative target sites in humanmRNAs (Supplementary Information S2).

In vitro identification of miRNA-targeting HPV-16Hsa-miR-139-3p identified as candidate miRNA. Of the 10 short-listed miRNAs, Hsa-miR-139-3p was significantly downregulated

in HPV-16–positive UPCI:SCC-090 (14-fold, P < 0.01), SiHa(122-fold, P < 0.01), and CaSki cell lines (19-fold, P < 0.01), andupregulated inHPV-negativeUPCI:SCC116 cells (4.2-fold) whencompared with primary culture (Fig. 1A; Supplementary Infor-mation S3). Furthermore, its expression was not affected byexternal factors including cell growth media (SupplementaryInformation S4).

In silico analysis revealed that Hsa-miR-139-3p had fourputative targets in E1 region of HPV-16 mRNA and no targetssites in E7 region (Fig. 1B; Supplementary Information S4).These observations were further validated by Luciferase Assayin which Hsa-miR-139-3p mimic transfected HPV-negative

Figure 1.

Hsa-miR-139-3p expression in HPV-16–positive and HPV-negative cell lines. A, Relative expression of Hsa-miR-139-3p in HPV-16-positive (UPCI:SCC090, SiHa, CaSki) cell lines, primary keratinocytes, and HPV-negative (UPCI:SCC 116) cells. B, Putative targets of Hsa-miR-139-3p in HPV-16 E1 mRNAregion. C, Relative FL repressive effect was analyzed using four FL plasmid constructs: pMiR as vector control, pMiR-E1 containing the putativesites, and pMiR-E7 as external vector control with viral DNA and no putative sites of Hsa-miR-139-3p. HPV-negative UPCI:SCC 116 cells werecotransfected with 1 mg of pMiR-plasmids and Renilla pRL-SV40P plasmid, and 100 pmol/L scrambled (S), Hsa-miR-139-3p mimic (M), andInhibitor (I). Renilla luciferase was used as an internal control to normalize FL activity expression. Results were compared with scrambled miRNAtransfected cells for statistical analyses. Plasmid containing the E1 region of primary HPV-16 mRNA showed significant decreased FL activity.� , P < 0.05; �� , P < 0.01.






UPCI:SCC-116 cells showed 30% to 35% decrease in FL activityin case of pMiR-E1 plasmid (P < 0.01) and 15% to 18%decrease in pMiR-E1þE7 (P < 0.01, 1:1) compared with control(Scrambled miRNA) and Hsa-miR-139-3p inhibitor–trans-fected cells (Fig. 1C).

Hsa-miR-139-3p overexpression/inhibition modulated HPVpathway genes

Hsa-miR-139-3p overexpression in HPV-16–positive UPCI:SCC-090 cells decreased HPV-16 mRNA (E6 and E7) levels (6.5-to 6.9-fold, P < 0.01; Fig. 2A). Moreover, Hsa-miR-139-3poverexpression in HPV-16–positive UPCI:SCC-090 cells alsoaffected expression of HPV pathway proteins in a dose-depen-dent manner (Fig. 2B and Supplementary Information S5). Hsa-miR-139-3p overexpression decreased expression of HPV-16 E6(1.8-fold, P < 0.05), p16 (5.5-fold, P < 0.05), and increased p53(20-fold, P < 0.05) and p21 expression (10.6 fold, P < 0.05) inHPV-16–positive cells (SiHa, UPCI:SCC-090, and CaSki) ascompared with control (scrambled miRNA-transfected HaCatand UPCI:SCC-116 cells; Fig. 2C–E). Hsa-miR-139-3p inhibitorshowed no significant changes.

Hsa-miR-139-3p overexpression/inhibition modulated hostoncogenic processes

Hsa-miR-139-3p overexpression decreased cell proliferationrate in HPV-positive cells (�49%, CaSki and �38%, SiHa) andits inhibition increased cell proliferation rate (14%, CaSki; 79%,SiHa). However, no significant change in proliferation wasobserved in HPV-negative cells (Fig. 3A).

Hsa-miR-139-3p overexpression resulted in decreased cell via-bility of HPV-16–positive cells (20%, SiHa; 27%, UPCI:SCC-090)and inhibition increased cell viability (35%, SiHa; 6%, UPCI:SCC-090; Fig. 3B).

Hsa-miR-139-3p overexpression increased accumulation ofcell at G2–M phase in HPV-16–positive cells (105%, UPCI:SCC-090 and 201%, SiHa) as compared with HPV-negative cells(7.2%, UPCI:SCC 116) and inhibitor transfected cells (13% to31%; Fig. 3C).

Hsa-miR-139-3p overexpression resulted in significantlyincreased cell death in case of HPV-16–positive cells (84.6%,UPCI:SCC-090; 55%, SiHa) when compared with HPV-negativecells (5.8%, UPCI:SCC-116; 7.3%, HaCat) and Hsa-miR-139-3pinhibition (Fig. 3D).

Rel

ativ

e ex

pre

ssio

n o

f H

PV-

16m

RN

As

(E6/

E7:

18S

)

0.0

HPV 16 E6

p53

Scr

amb

led

miR

NA

tra

nsf

ecti

on

Hsa

-miR

-139

-3p

mim

ic t

ran

sfec

tio

n

Hsa

-miR

-139

-3p

inh

ibit

or

tran

sfec

tio

n

p16

p21

GAPDH

HPV 16 E6

p53

p16

p21

GAPDH

HPV 16 E6

p53

p16

p21

GAPDH

HPV Negative HPV Positive

HPV 16 E6

p16

p21

p53

GAPDH

UPCI:SCC 116 UPCI:SCC 090

100 pmol/L Hsa-miR-139-3pmimic/inhibitor

Scram

bled_E

7

HaCat

SCC-116

SCC-090

CaSki

SiHa

HaCat

SCC-116

SCC-090

CaSki

SiHa HaC

at

SCC-116

SCC-090

CaSki

SiHa

Mimic_

E7

Inhib

itor_

E7

Scram

bled_E

6

Scrambled

+ +

++

++

–

– – – –

– ––

– – ––

100 pmol/L Mimic

200 pmol/L Mimic

Mimic_

E6

Inhib

itor_

E6

0.6

1.2

1.8

A

C D E

B

Figure 2.

Hsa-miR-139-3p overexpression/inhibition leads to significant changes in expression of targeted genes in HPV-positive cells. A, Relative expression ofHPV16 mRNA (E6 and E7 primers) in UPCI:SCC 090 cells transfected with 100 pmol Hsa-miR-139-3p mimic (Mimic_E7 and E6) compared with scrambledcontrol (Scrambled_E7 and E6) and 100 pmol Hsa-miR-139-3p Inhibitor (Inhibitor_E7 and E6). �� , P < 0.01. B, Western blot analysis of HPV-16 E6 proteins andHPV-targeted pathway proteins: p53, p16, and p21 in HPV-negative UPCI:SCC 116 and HPV-positive UPCI:SCC 090 cells transfected with 100 and200 pmol Hsa-139-3p Mimic. C, Western blot analysis in HPV-negative cells (HaCat, UPCI:SCC 116) and HPV-16–positive cells (SiHa, UPCI:SCC 116, CaSki)transfected with 100 pmol of scrambled miRNA, (D) Hsa-miR-139-3p mimic, and (E) Hsa-miR-139-3p inhibitor.

Sannigrahi et al.





Hsa-miR-139-3p overexpression significantly decreased cellmigration as longer time interval for gap closure was observedinHPV-16–positive cells as compared either scrambledmiRNAormiRNA inhibitor or Hsa-miR-139-3p overexpressed HPV-nega-tive cells (Fig. 3E).

These results suggest Hsa-miR-139-3p acts as antiviral miRNA,restricting HPV-16 oncoprotein expression (E6, E7), recovers

tumor-suppressors (p53, p21), and resulting in decreased cellproliferation, G2–M arrest, and cell death in HPV-16–infectedcells.

Ex vivo analysis of Hsa-miR-139-3p expressionHsa-miR-139-3p expression downregulated in HPV-positive tissuesamples. Hsa-miR-139-3p expression was also found to be

Figure 3.

Overexpression and inhibition of Hsa-miR-139-3p in HPV-16–positive and HPV-negative cells. HPV-negative cells (UPCI:SCC 116 and HaCat) andHPV-16–positive cells (UPCI:SCC 090, SiHa, and CaSki) transfected with 100 pmol scrambled/mimic/inhibitor. A, Graph shows EdU stained cellsafter FACS analysis. B, Graphs show the average viability of cells in MTT cell viability assay relative to the appropriate scrambled control (taken as 100%).C, Graphs show FACS analysis of pI stained transfected with 100 pmol of Hsa-miR-139-3p mimic compared with scrambled transfected cells. D,Graph showing average percentage of dying cells. E, Graph shows percentage of space between two monolayes after scratch assay at differenttime intervals. HPV-16 positive. �, P < 0.05.






significantly downregulated in HPV-16–positive HNC (n ¼ 27)and HPV-positive cervical cancer (n ¼ 254) with respect to HPV-negative samples (n ¼ 238) from TCGA (Fig. 4A).

In our HNC cohort (n¼ 110), Hsa-miR-139-3p expression wasdownregulated 12-fold in HPV-16 DNA positive HNC (n ¼ 30)with respect to HPV-negative HNC tissue samples (n ¼ 40, P <0.001) and 21-fold with respect to control (HPV-negative non-cancerous) tissue samples (n ¼ 40, P < 0.001; Fig. 4B). Out of30 HPV 16 DNA-positive samples, 20 (66.7%) showed HPV-16E7-mRNA amplification suggesting an active infection. Hsa-miR-139-3p expression was also significantly downregulated (P <0.05) in E7 mRNA–positive HNC tissues (n ¼ 20) as comparedwith E7 mRNA negative HNC tissues (Fig. 4C). We also observedthat E7 mRNA expression is negatively correlated with Hsa-miR-139-3p expression [r ¼ �0.07, P ¼ 0.4; 95% confidence interval(CI),�0.5–0.4; Fig. 4D). Furthermore, HPV-16 E7–positive HNCtissues (n¼ 20) had significantly higher viral load and integratedform of viral DNA. The viral load ranged from <1 to 537.6 � 103

copies per 103 cells. Hsa-miR-139-3p expression had significantnegative co-relation with viral load, that is higher viral loadhad lower Hsa-miR-139-3p expression and vice-versa (r ¼�0.42, P ¼ 0.03; 95% CI, �0.73–0.04; Fig. 4D). Hsa-miR-139-3p expression was also decreased in p16 IHC positive (n ¼ 14),p53-negative (n ¼ 10), and/or pRB-negative (n ¼ 13) biopsysamples (Supplementary Information S4).

These result suggest decreased Hsa-miR-139-3p expression co-related with active HPV infection.

TCGA survival data correlated decreased Hsa-miR-139-3p expres-sion with active HPV infection. Cervical cancer patients are 99%HPV-positive with poor survival rate (34). However, we observedcervical cancer cases with increased Hsa-miR-139-3p expression(high, n ¼ 82, relative-expression >5.93) had significantly bettersurvival (HR 0.7; 95% CI, 0.4–1.2; P < 0.05) than those havingdecreased Hsa-miR-139-3p expression (low, n ¼ 136, relative-expression <5.93; Fig. 5A).

HPV-16–positive HNC have significantly better survival(HR 0.4; 95% CI, 0.2–0.8; P < 0.01) than HPV-negativeHNC (P < 0.01; Fig. 5B). However, HPV16-positive HNCwith increased Hsa-miR-139-3p expression (high, n ¼ 9,relative-expression >15.04) had better survival (HR 0.8;95% CI, 0.07–8.3; P > 0.05) than those having decreasedHsa-miR-139-3p expression (low, n ¼ 12, relative-expression<15.04; Fig. 5C).

Regulation of expression of Hsa-miR-139-3p inHPV-positive HNC

In silico analysis revealed Hsa-miR-139-3p lies within theintronic region of PDE2A (Chromosome 11; 72615130-72615063). Further from TCGA data, PDE2A expressionshowed significant co-relation with Hsa-miR-139-3p expres-sion in cervical cancer (n ¼ 153) and HNC (n ¼ 249), suggest-ing both may be coordinately regulated (Fig. 6A). MethHC datashow a negative correlation between PDE2A expression andpromoter/CpG methylation in HNC and cervical cancer

Figure 4.

Relative expression of Hsa-miR-139-3p inHPV-16–positive HNSCC tissue samples.A, Relative expression of Hsa-miR-139-3pfrom TCGA data in HPV-negative HNC(n¼ 243), HPV-16–positive HNC (n¼ 29),and HPV-positive CC (n ¼ 254). B,Relative expression of Hsa-miR-139-3p inHPV-16 DNA-positive HNSCC (n ¼ 30),HPV-negative HNSCC (n ¼ 40), and OralHealth controls (n ¼ 40) biopsy samples.C, Relative expression of Hsa-miR-139-3pin HPV-16–positive E7 mRNA-positivesamples (n ¼ 20). D, Corelation of Hsa-miR-139-3p expression, viral load, and E7mRNA expression in HPV-16–positivesamples (n¼ 20). � , P < 0.05; �� , P < 0.01;�� , P < 0.001.

Sannigrahi et al.





(Supplementary Information S7). Comparing the methylationlevels from human pan-cancer methylation database, MethHCacross gene regions around promoter and CpG islands showedsignificantly increased methylation in HPV-16–positive HNC(n ¼ 29) and cervical cancer (n ¼ 175) with respect to HPV-negative (n ¼ 220) cases (Fig. 6B).

Further, 5-Aza-dC treatment of HPV-16–positive and HPV-negative cell-lines was carried out to examine if it had effecton Hsa-miR-139-3p expression. 5-Aza-dC treated HPV-16–positive UPCI:SCC-090 cells showed significantly increasedHsa-miR-139-3p expression (Fig. 6C; Supplementary Informa-tion S8).

Promoter/CpG methylation of PDE2A (Chromosome 11;72676856-72677047) was analyzed using methylation sen-sitive PCR (MSP; Supplementary Information S8). HPV-16–positive cells (UPCI:SCC-090, CaSki, and SiHa) showedsignificantly increased methylation as compared withHPV-negative (HEK-293, HaCat, and UPCI:SCC 116) celllines, which was validated by sequencing (Fig. 6D). MSPanalysis of HNC tissue samples showed that 36% (9/25) ofHPV-16–positive tissue samples showed promoter methyl-ation as compared with 7.6% (2/26) HPV-negative HNC

tissues and controls (HPV negative noncancerous; 2/26;Fig. 6E).

Analysis of HPV-16 integration at promoter site of PDE2Adid not reveal any integration of HPV-16 genome with PDE2A(Supplementary Information S8). Further role of histone acet-ylation on Hsa-miR-139-3p expression was measured by TSAtreatment of HPV-16–positive cell lines (Fig. 6C) and by ChIPin cell lines (Fig. 6E). However, no significant change in Hsa-miR-139-3p expression was observed (Supplementary Infor-mation S8).

Thus, our results suggest that promoter/CpG methylation reg-ulates Hsa-miR-139-3p expression in HPV-16–positive epithelialcell lines and HNSCC biopsy samples.

Hsa-miR-139-3p overexpression loweredchemotherapeutic dosage

Systemic treatment of HNC involves combination of cis-platin (CDDP) and 5-fluorouracil (5-FU) (35). Initially,LD50/IC50 values of CDDPþ5-FU (1:1) was determined inHPV-positive and HPV-negative cell lines. HPV-16–positivecell lines (UPCI:SCC-090, 31.34 mmol/L and SiHa, 26.65mmol/L) were observed to be more resistant to treatment as

Survival curve: Survival proportions in CCwith high and low Hsa-miR-139-3p expression

Survival curve :Survival proportions in HNCwith high and low Hsa-miR-139-3p expression

Survival curve :HPV-16+ve HNC vs HPV -ve HNC

100

A

C

B

75

50

25

0 0

50

100

150

0 2,000

050

60

70

80

90

Per

cen

t su

rviv

al

100

110

2,000

Low_HPV-16

High_HPV-16

3,0001,000

4,000

Hsa-miR-139 HighHPV16+ve HNC

HPV-ve HNCHsa-miR-139 Low

Days

Days

Per

cen

t su

rviv

al

Per

cen

t su

rviv

al

6,000 8,000 1,000 2,000

Time (days)

3,000 4,000 5,0000

Figure 5.

Survival analysis in HNC and cervical cancer tissue samples from TCGA data. Kaplan–Meier survival curves of (A) HPV-positive cervical cancer patientswith high Hsa-miR-139-3p expression (n ¼ 82) compared with low Hsa-miR-139-3p expression (n ¼ 136). (B) HPV-16–positive HNC patients (n ¼ 21)compared with HPV-negative HNC patients (n ¼ 192). C, Low Hsa-miR-139-3p expressed HPV16–positive HNC patients (n ¼ 12) compared with HighHsa-miR-139-3p expressed HPV16–positive HNC patients (n ¼ 9). � , P < 0.05; �� , P < 0.01.






compared with HPV-negative cell lines (UPCI:SCC-116, 8.626mmol/L and HaCat, 8.894 mmol/L; Fig. 7A; SupplementaryInformation S9).

Further, on Hsa-miR-139-3p overexpression followed bytreatment of chemotherapeutic drugs (5 to 20 mmol/L) resultedin a 20% to 50% decrease in LD50 values (Fig. 7B–E) in HPV-positive cells (SiHa, 11.16 mmol/L and UPCI:SCC-090, 11.06mmol/L) cells as compared with scrambled miRNA-transfectedHPV-16–positive cells (SiHa, 18.61 mmol/L and UPCI:SCC-090, 16.63 mmol/L) and Hsa-miR-139-3p overexpressedHPV-16–negative cells (HaCat, 17.08 mmol/L and UPCI:SCC-116, 14.60 mmol/L), suggesting Hsa-miR-139-3p overexpres-sion sensitizes cells to a CDDPþ5-FU treatment in HPV-16–positive cell lines.

DiscussionRecent reports suggest that miRNAs play an important role in

host–viral interactions. In this study, we have addressed howHPVmay modulate host antiviral miRNAs to their own benefit. Weidentified Hsa-miR-139-3p, a host miRNA having putative targeton HPV-16 mRNA. Our results suggest that HPV-16 downregu-lates Hsa-miR-139-3p expression to create a favorable environ-ment for viral replication and oncogenesis.

Although several miRNAs showed binding affinity for HPV-16mRNA, only Hsa-miR-139-3p expression was significantlydecreased in HPV-16–positive HNC tissues and cell lines. Thisis further supported by TCGA data, where Hsa-miR-139-3p wasfound significantly downregulated in HPV-positive HNC and

0

–0.2

TCGA Tissue samplesHPV-ve

_HNC P

rom

oter

HPV16+v

e_HNC P

rom

oter

CC_Pro

mote

r

HPV-ve

_HNC C

pG

HPV-16

+ve_

CpG

CC_CpG

–0.1

0.0

0.1

0.2

0.3

0.4

0200400600800

1,000

0

0

Cell lines Cell lines Cell linesDMSO_S

CC 116

AZA_116

TSA_116

DMSO_SCC09

0

AZA_090

TSA_090

HPV Neg

ative

HPV Posit

ive

HaCat

_H3

SCC116_

H3

SCC090_

H3

CaSki_

H3

HaCat

_H4

SCC116_

H4

SCC090_

H4

CaSki_

H4

INPUT

2

0

0

0.0

0.5

1.0

2

4

Inte

nsi

ty o

f m

ethy

lati

on

Rel

ativ

e ex

pre

ssio

n(m

iR-1

39-3

p: U

6)

6

8

4

Rel

ativ

e ex

pre

ssio

n(m

iR-1

39-3

p: U

6)

6

10 20 30 40

Hsa-miR-139-3p Expression

Hsa-miR-139-3p Expression

50 100 150

P = 0.000***

P = 0.000***

200

1,000

2,000

3,000

4,000

A B

C D E

PD

E2A

Exp

ress

ion

Tum

or

met

hyla

tio

nat

PD

E2A

pro

mo

ter

PD

E2A

Exp

ress

ion

Correlation in Head and Neck Cancer

Correlation in Cervical Cancer

Figure 6.

Methylation and ChIP assays in cell lines and biopsy samples. A, Comparison of PDE2A expression and Hsa-miR-139-3p expression by linear regression analysisin HNC (n ¼ 264) and cervical cancer (n ¼ 153) cases from MethHC. B, Methylation levels of Hsa-miR-139-3p harboring gene PDE2A at promoter andCpG island region in HPV-16–positive (n ¼ 29), HPV-negative (n ¼ 236) HNC, and cervical cancer (n ¼ 172) cases. C, Relative expression of Hsa-miR-139-3pin cells treated with 5-aza-dC concentrations (10 mmol/L) and TSA (100 ng/mL) for 4 days. D, HPV-16–positive cell lines (UPCI:SCC-090, SiHa, andCaSki) and HPV-negative cell lines (HEK, HaCat, and UPCI:SCC-116) amplification band intensities using methylated and unmethylated-specific primerswere analyzed using ImageJ software. E, Relative expression of Hsa-miR-139-3p in HPV-16–positive (UPCI:SCC 090, CaSki) and HPV-negative (HaCat, UPCI:SCC 116) cell lines in ChIP. � , P < 0.05; �� , P < 0.01; �� , P < 0.001.

Sannigrahi et al.





cervical cancer cases. It is also reported to be dysregulated incervical cancer (36), colon cancer (37), bladder cancer (38), andleukemia (39).

Hsa-miR-139-3p was found to target E1 region (between1703 and 2044) of HPV-16 by luciferase reporter assay. Studiesshowed that E1 expression is critical for and limiting in theinitial amplification of the HPV-16 genome (40) and also formaintenance replication during keratinocyte differentiation(41). In accordance with these studies, a negative correlationwas observed between HPV-16 viral load and Hsa-miR-139-3pexpression in HNC tissue samples, suggesting potential role of

Hsa-miR-139-3p in regulating HPV-16 replication during theinitiation of infection. Further, this region (between 1703 and2044) is present in most fusion transcripts including early genetranscript from episomal HPV (E7-E1^E4) and several lategene transcripts from integrated HPV (such as E7-E1^cellularRNA, E7-E1^E4-cellular RNA, etc.) in HPV-16–infected tissues(42, 43). Thus, in those cases where HPV-16 has integrated intogenome, Hsa-miR-139-3p may also target late HPV-16 mRNAswhich in turn is known to regulates the expression of otheroncogenic proteins like E6 and E7 and thereby regulate onco-genesis. In accordance to these studies, we observed a decreased

Figure 7.

Dose–response curve analysis of inhibition of cell growth by the drug treatment correlated to drug concentrations and corresponding IC50/LD50 values.A, Cells were treated for 48 hours with various concentrations of combination of CDDP and 5-FU from 1 to 100 mmol/L (1, 2, 4, 8, 10, 20, 40, 80, and 100)to determine the IC50 or LD50 value for the combination treatment. B–E, Cells were transfected with 50 pmol Hsa-miR-139-3p mimic/inhibitor. After24 hours, cells were treated with 5, 10, and 20 mmol/L concentration of cisplatin and 5-FU combination for 48 hours.






relative E7 mRNA expression co-related with high Hsa-miR-139-3p expression in biopsy samples and vice-versa. To furtherexamine its role, Hsa-miR-139-3p was overexpressed in HPV-16–positive cell lines and this resulted in suppression of E6and E7 which induced revival of tumor suppressors: p53 andp21, indicating its role in restricting HPV-16 expression. Sim-ilar results were observed in in vitro and in vivo studies wheresiRNAs used to directly silence E6/E7 expression (42–44),suggesting Hsa-miR-139-3p regulates E6/E7 expression. More-over, Hsa-miR-139-3p overexpression also decreased cellgrowth/proliferation and increased cell death. This furthervalidated antiviral role of Hsa-miR-139-3p as revival of p53/p21 pathway in viral-infected cells is known to mediategrowth arrest, apoptosis, and necrosis (45, 46). Thus, Hsa-miR-139-3p acts as antiviral miRNA by limiting expression ofHPV-16 oncogenes.

To investigate whether HPV-16 is directly modulating Hsa-miR-139-3p, an ex vivo analysis was done in HPV-16–positiveHNC tissues samples which showed decreased Hsa-miR-139-3pexpression correlated with increased HPV-16 viral load andactive viral infection (HPV-16-E7 mRNA expression), suggest-ing HPV-16 may be downregulating Hsa-miR-139-3p expres-sion. To further understand the mechanisms behind down-regulation of Hsa-miR-139-3p, epigenetic mechanisms likehistone acetylation and CpG/promoter methylation wereexamined. In human genome, pre-miR-139 (that encodesmature miR-139-3p) is located on the human chromosome11q13.4 region NC_000011.10 (72615063. . .72615130, com-plement) and this miRNA is encoded within the second intronof phosphodiesterase 2A (PDE2A) gene. Hsa-miR-139-3pexpression correlated with expression of its harboring genePDE2A in cervical cancer and HNC samples. While examiningmethylation status of PDE2A promoter/CpG islands, weobserved a significantly increased methylation level at its pro-moter/CpG regions in HNC tissues by MSP. Increased tumormethylation levels were also observed in HPV-positive HNCand CC cases fromMethHC database indicating promoter/CpGmethylation decreases Hsa-miR-139-3p expression. As HPV-16is known to induce DNA methylation pathway to activate thedevelopment of cancer (47, 48), it is evident that downregula-tion of Hsa-miR-139-3p that targets HPV-mRNAs by promotermethylation may be a survival mechanism adopted by HPV-16to create a more favorable environment for its replication.However, studies on host cellular proteins targeted by Hsa-miR-139-3p and other host miRNAs during such host–viralinteraction that may also play important role in these survivalmechanisms adopted by HPV-16 leading to oncogenesis arealso warranted.

Survival analysis using TCGA data showed HPV-16–positivepatients with increased Hsa-miR-139-3p expression has bettersurvival (HR <1) than those with decreased Hsa-miR-139-3pexpression in HPV-16–positive patients HNC and cervicalcancer patients suggesting high Hsa-miR-139-3p expression isrestricting HPV-16 expression which is in-turn limiting itsoncogenesis.

Because Hsa-miR-139-3p was found to promote apoptosis ofHPV positive cells, we investigated if it could potentiate responseto chemotherapy asmicroRNAs have been reported to be effectiveas adjunct therapy to chemo-/radiotherapy-resistant cells (49).We found that HPV-16–positive cells had higher LD50/IC50

values, probably due to degradation/inhibition of p53/p21 byHPV oncogenes as chemotherapy drugs, CDDP and 5-FU areknown to act through p53-mediated apoptotic pathways (50,51). However, Hsa-miR-139-3p overexpression in HPV-16–pos-itive cells resulted in decreased LD50/IC50 values by 20% to 50%,indicating that Hsa-miR-139-3p was sensitizing these cells tochemotherapy. Thus, our results suggest that Hsa-miR-139-3pcould be a potential adjunct therapy in chemotherapeutic treat-ment of HPV-16–infected patients.

In summary, our work onHsa-miR-139-3p adds to the growingevidence of association between microRNAs and HPV in cancers.Thus, we propose a model of HPV infection where-in down-regulation of Hsa-miR-139-3p by promoter methylation in HPV-16–infected cells results in upregulation of pro-oncogenic path-ways leading to HPV-16–induced carcinogenesis. However,because HPV-16 are known to multiply in basal epithelium layerand cell line model with high expression of HPV-16 oncogenes(E6 and E7) and not expressing HPV-16 E1 may not reflect anormal HPV infection cycle during epidermal differentiation,hence studies on transgenic mouse model and 3D cell culturemodels for defining its prognostic implications are warranted.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: M.K. Sannigrahi, R. Sharma, N.K. Panda, V. Rattan,M. KhullarDevelopment of methodology: M.K. Sannigrahi, R. Sharma, M. KhullarAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M.K. Sannigrahi, V. Singh, N.K. Panda, V. RattanAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M.K. Sannigrahi, R. SharmaWriting, review, and/or revision of the manuscript: M.K. Sannigrahi,R. Sharma, M. KhullarAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M.K. Sannigrahi, M. KhullarStudy supervision: R. Sharma, N.K. Panda, V. Rattan, M. Khullar

AcknowledgmentsThis study was carried out at the Genetics of Complex Disorder laboratory,

PGIMER, Chandigarh, India. UPCI:SCC-116 and UPCI:SCC-090 cell lines werekind gifts from Dr. S. Gollin (University of Pittsburgh, Pittsburgh, PA). Theauthors are grateful to all individuals who participated in this study and thelaboratory staff members for their skillful technical help. M.K. Sannigrahithankfully acknowledges research fellowship from Indian Council of MedicalResearch, New Delhi, India.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received November 24, 2016; revised January 10, 2017; accepted January 11,2017; published OnlineFirst January 31, 2017.

References1. Doorbar J.The papillomavirus life cycle. J Clin Virol 2005;32:S7–15.2. Stubenrauch F, Laimins LA. Human papillomavirus life cycle: active and

latent phases. Semin Cancer Biol 1999;9:379–86.

3. Steenbergen RD, Snijders PJ, Heideman DA, Meijer CJ. Clinical implica-tions of (epi)genetic changes in HPV-induced cervical precancerouslesions. Nat Rev Cancer 2014;14:395–405.

Sannigrahi et al.





4. MoodyCA, Laimins LA.Humanpapillomavirus oncoproteins: pathways totransformation. Nat Rev Cancer 2010;10:550–60.

5. Scaria V, Hariharan M, Pillai B, Maiti S, Brahmachari SK. Host-virusgenome interactions: macro roles for microRNAs. Cell Microbiol 2007;9:2784–94.

6. Huang J, Wang F, Argyris E, Chen K, Liang Z, Tian H, et al. CellularmicroRNAs contribute to HIV-1 latency in resting primary CD4þ T lym-phocytes. Nat Med 2007;13:1241–7.

7. Lecellier CH, Dunoyer P, Arar K, Lehmann-Che J, Eyquem S, Himber C,et al. A cellular microRNA mediates antiviral defense in human cells.Science 2005;308:557–60.

8. Otsuka M, Jing Q, Georgel P, New L, Chen J, Mols J, et al. Hypersuscep-tibility to vesicular stomatitis virus infection inDicer1-deficientmice is dueto impaired miR24 and miR93 expression. Immunity 2007;27:123–34.

9. Navratil V, de Chassey B, Combe CR, Lotteau V. When the human viralinfectome and diseasome networks collide: towards a systems biologyplatform for the aetiology of human diseases. BMC Syst Biol 2011;5:13.

10. Skalsky RL, Cullen BR. Viruses, microRNAs, and host interactions. Ann RevMicrobiol 2010;64:123–41.

11. Mahajan VS, Drake A, Chen J. Virus-specific host miRNAs: antiviraldefenses or promoters of persistent infection? Trends Immunol 2009;30:1–7.

12. Kakumani PK, Ponia SS, S RK, Sood V, Chinnappan M, Banerjea AC, et al.Role of RNA interference (RNAi) in dengue virus replication and identi-fication of NS4B as an RNAi suppressor. J Virol 2013;87:8870–83.

13. Matskevich AA, Moelling K. Dicer is involved in protection against influ-enza A virus infection. J Gen Virol 2007;88:2627–35.

14. de Vries W, Haasnoot J, van der Velden J, van Montfort T, Zorgdrager F,PaxtonW, et al. Increased virus replication inmammalian cells by blockingintracellular innate defense responses. Gene Ther 2008;15:545–52.

15. Sullivan CS.New roles for large and small viral RNAs in evading hostdefences. Nat Rev Genet 2008;9:503–7.

16. Cameron JE, Fewell C, Yin Q, McBride J, Wang X, Lin Z, et al. Epstein-Barrvirus growth/latency III program alters cellular microRNA expression.Virology 2008;382:257–66.

17. Lee JW, Choi CH, Choi JJ, Park YA, Kim SJ, Hwang SY, et al. AlteredMicroRNA expression in cervical carcinomas. Clin Cancer Res 2008;14:2535–42.

18. Wald AI, Hoskins EE, Wells SI, Ferris RL, Khan SA. Alteration of microRNAprofiles in squamous cell carcinoma of the head and neck cell lines byhuman papillomavirus. Head Neck 2011;33:504–12.

19. Jimenez-Wences H, Peralta-Zaragoza O, Fernandez-Tilapa G. Humanpapilloma virus, DNA methylation and microRNA expression in cervicalcancer (Review). Oncol Rep 2014;31:2467–76.

20. White JS, Weissfeld JL, Ragin CC, Rossie KM, Martin CL, Shuster M, et al.The influence of clinical and demographic risk factors on the establishmentof head and neck squamous cell carcinoma cell lines. Oral Oncol 2007;43:701–12.

21. Sannigrahi MK, Singh V, Sharma R, Panda NK, Radotra BD, Khullar M.Detection of active human papilloma virus-16 in head and neck cancers ofAsian North Indian patients. Oral Dis 2016;22:62–8.

22. Rusinov V, Baev V, Minkov IN, Tabler M. MicroInspector: a web tool fordetection of miRNA binding sites in an RNA sequence. Nucleic Acids Res2005;33:W696–700.

23. Chang TH, Huang HY, Hsu JB, Weng SL, Horng JT, Huang HD. Anenhanced computational platform for investigating the roles of regulatoryRNA and for identifying functional RNA motifs. BMC Bioinformatics2013;14:S4.

24. Rehmsmeier M, Steffen P, Hochsmann M, Giegerich R. Fast and effectiveprediction of microRNA/target duplexes. RNA 2004;10:1507–17.

25. Garcia DM, Baek D, Shin C, Bell GW, Grimson A, Bartel DP. Weak seed-pairing stability and high target-site abundance decrease the proficiency oflsy-6 and other microRNAs. Nat Struct Mol Biol 2011;18:1139–46.

26. Hampf M, Gossen M. A protocol for combined Photinus and Renillaluciferase quantification compatible with protein assays. Anal Biochem2006;356:94–9.

27. Jung HM, Phillips BL, Chan EK. miR-375 activates p21 and suppressestelomerase activity by coordinately regulating HPV E6/E7, E6AP, CIP2A,and 14–3-3zeta. Mol Cancer 2014;13:80.

28. ChenX,Prywes R. Serum-induced expressionof the cdc25Ageneby relief ofE2F-mediated repression. Mol Cell Biol 1999;19:4695–702.

29. Salic A, Mitchison TJ. A chemical method for fast and sensitive detection ofDNA synthesis invivo. Proc Nat Acad Sci U S A 2008;105:2415–20.

30. Liang CC, Park AY, Guan JL. Invitro scratch assay: a convenient andinexpensive method for analysis of cell migration invitro. Nat Protoc2007;2:329–33.

31. Comprehensive genomic characterization of head and neck squamous cellcarcinomas. Nature 2015;517:576–82.

32. Parfenov M, Pedamallu CS, Gehlenborg N, Freeman SS, Danilova L,Bristow CA, et al. Characterization of HPV and host genome interactionsin primary head and neck cancers. Proc Natl Acad Sci U S A 2014;111:15544–9.

33. Huang WY, Hsu SD, Huang HY, Sun YM, Chou CH, Weng SL, et al.MethHC: a database of DNA methylation and gene expression in humancancer. Nucleic Acids Res 2015;43:D856–61.

34. Clifford GM, Smith JS, Aguado T, Franceschi S. Comparison of HPV typedistribution in high-grade cervical lesions and cervical cancer: a meta-analysis. Br J Cancer 2003;89:101–5.

35. Seiwert TY, Salama JK, Vokes EE. The chemoradiation paradigm in headand neck cancer. Nat Clin Pract Oncol 2007;4:156–71.

36. Lajer CB, Garnaes E, Friis-Hansen L, Norrild B, Therkildsen MH, Glud M,et al. The role of miRNAs in human papilloma virus (HPV)-associatedcancers: bridging between HPV-related head and neck cancer and cervicalcancer. Br J Cancer 2012;106:1526–34.

37. Liu X, Duan B, Dong Y, He C, Zhou H, Sheng H, et al. MicroRNA-139–3pindicates a poor prognosis of colon cancer. Int J Clin Exp Pathol2014;7:8046–52.

38. Jia AY, Castillo-Martin M, Domingo-Domenech J, Bonal DM, Sanchez-Carbayo M, Silva JM, et al. A common microRNA signature consisting ofmiR-133a, miR-139-3p, and miR-142-3p clusters bladder carcinoma insitu with normal umbrella cells. Am J Pathol 2013;182:1171–9.

39. Alemdehy MF, Haanstra JR, de Looper HW, van Strien PM, Verhagen-Oldenampsen J, Caljouw Y, et al. ICL-induced miR139-3p and miR199a-3p have opposite roles in hematopoietic cell expansion and leukemictransformation. Blood 2015;125:3937–48.

40. Lace MJ, Anson JR, Turek LP, Haugen TH. Functional mapping of thehuman papillomavirus type 16 E1 cistron. J Virol 2008;82:10724–34.

41. EgawaN,Nakahara T, Ohno S, Narisawa-SaitoM, Yugawa T, FujitaM, et al.The E1 protein of human papillomavirus type 16 is dispensable formaintenance replication of the viral genome. J Virol 2012;86:3276–83.

42. Jonson AL, Rogers LM, Ramakrishnan S, Downs LSJr.Gene silencing withsiRNA targeting E6/E7 as a therapeutic intervention in a mouse model ofcervical cancer. Gynecolo Oncol 2008;111:356–64.

43. Sima N, Wang W, Kong D, Deng D, Xu Q, Zhou J, et al. RNA interferenceagainstHPV16 E7 oncogene leads to viral E6 and E7 suppression in cervicalcancer cells and apoptosis via upregulation of Rb and p53. Apoptosis2008;13:273–81.

44. Yoshinouchi M, Yamada T, Kizaki M, Fen J, Koseki T, Ikeda Y, et al. Invitroand invivo growth suppression of human papillomavirus 16-positivecervical cancer cells by E6 siRNA. Mol Ther 2003;8:762–8.

45. Munagala R, Aqil F, Jeyabalan J, Gupta RC. Tanshinone IIA inhibits viraloncogene expression leading to apoptosis and inhibition of cervical cancer.Cancer Lett 2015;356:536–46.

46. Wang Y, Yuan Z, You C, Han J, Li H, Zhang Z, et al. Overexpressionp21WAF1/CIP1 in suppressing retinal pigment epithelial cells and pro-gression of proliferative vitreoretinopathy via inhibition CDK2 and cyclinE. BMC Ophthalmol 2014;14:144.

47. Li L, Xu C, Long J, ShenD, ZhouW, ZhouQ, et al. E6 and E7 gene silencingresults in decreased methylation of tumor suppressor genes and inducesphenotype transformation of human cervical carcinoma cell lines. Onco-target 2015;6:23930–43.

48. Zhang HD, Jiang LH, Sun DW, Li J, Tang JH. MiR-139-5p: promisingbiomarker for cancer. Tumour Biol 2015;36:1355–65.

49. Hwang JH, Voortman J, Giovannetti E, Steinberg SM, Leon LG, Kim YT,et al. Identification of microRNA-21 as a biomarker for chemoresistanceand clinical outcome following adjuvant therapy in resectable pancreaticcancer. PLoS One 2010;5:e10630.

50. Niedner H, Christen R, Lin X, Kondo A, Howell SB. Identification of genesthat mediate sensitivity to cisplatin. Mol Pharmacol 2001;60:1153–60.

51. Petak I, Tillman DM, Houghton JA. p53 dependence of Fas inductionand acute apoptosis in response to 5-fluorouracil-leucovorin in humancolon carcinoma cell lines. Clin Cancer Res 2000;6:4432–41.






2017;23:3884-3895. Published OnlineFirst January 31, 2017.Clin Cancer Res M.K. Sannigrahi, Rajni Sharma, Varinder Singh, et al. Carcinomas

Induced−Role of Host miRNA Hsa-miR-139-3p in HPV-16

Updated version

10.1158/1078-0432.CCR-16-2936doi:

Access the most recent version of this article at:

Material

Supplementary

http://clincancerres.aacrjournals.org/content/suppl/2017/01/31/1078-0432.CCR-16-2936.DC1

Access the most recent supplemental material at:

Cited articles

http://clincancerres.aacrjournals.org/content/23/14/3884.full#ref-list-1

This article cites 51 articles, 12 of which you can access for free at:

Citing articles

http://clincancerres.aacrjournals.org/content/23/14/3884.full#related-urls

This article has been cited by 2 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://clincancerres.aacrjournals.org/content/23/14/3884To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/lookup/doi/10.1158/1078-0432.CCR-16-2936

http://clincancerres.aacrjournals.org/content/suppl/2017/01/31/1078-0432.CCR-16-2936.DC1

http://clincancerres.aacrjournals.org/content/23/14/3884.full#ref-list-1

http://clincancerres.aacrjournals.org/content/23/14/3884.full#related-urls

http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://clincancerres.aacrjournals.org/content/23/14/3884


role of host mirna hsa-mir-139-3p in hpv-16 induced …biology of human tumors role of host mirna...

Documents