research article identification of circulating...

Research ArticleIdentification of Circulating miRNAs in a Mouse Model ofNerve Allograft Transplantation under FK506Immunosuppression by Illumina Small RNA Deep Sequencing

Shao-Chun Wu,1 Cheng-Shyuan Rau,2 Johnson Chia-Shen Yang,3

Tsu-Hsiang Lu,3 Yi-Chan Wu,3 Yi-Chun Chen,3 Siou-Ling Tzeng,3

Chia-Jung Wu,3 Chia-Wei Lin,3 and Ching-Hua Hsieh3

1Department of Anesthesiology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine,No. 123, Ta-Pei Road, Niao-Song District, Kaohsiung 833, Taiwan2Department of Neurosurgery, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine,No. 123, Ta-Pei Road, Niao-Song District, Kaohsiung 833, Taiwan3Department of Plastic and Reconstructive Surgery, Kaohsiung Chang Gung Memorial Hospital andChang Gung University College of Medicine, No. 123, Ta-Pei Road, Niao-Song District, Kaohsiung 833, Taiwan

Correspondence should be addressed to Ching-Hua Hsieh; [email protected]

Received 16 April 2015; Accepted 8 July 2015

Academic Editor: Mariann Harangi

Copyright © 2015 Shao-Chun Wu et al.This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background.This study aimed to establish the expression profile of circulatingmicroRNAs (miRNAs) during nerve allotransplanta-tion in the presence and absence of FK506 immunosuppression.Methods. A 1 cmBALB/c donor sciatic nerve graftwas transplantedinto the sciatic nerve gaps created in recipient C57BL/6mice with or without daily FK506 immunosuppression [1mg/(kg⋅d)]. At 3, 7,and 14 d after nerve allotransplantation, serum samples were collected for miRNA expression analysis by Illumina small RNA deepsequencing. Results. Sequence analysis showed that the dominant size of circulating small RNAs after nerve allotransplantation was22 nucleotides, followed by 23-nucleotide sequences. Nine upregulated circulating miRNAs (let-7e-5p, miR-101a-3p, miR-151-5p,miR-181a-5p, miR-204-5p, miR-340-5p, miR-381-3p, miR-411-5p, miR-9-5p, and miR-219-2-3p) were identified at 3 d, but none wasidentified at 7 or 14 d. Among them, miR-9-5p had the highest fold-change of >50-fold, followed bymiR-340-5p with 38.8-fold.Thepresence of these ninemiRNAswas not significant at 7 and 14 d after nerve allotransplantationwith orwithout immunosuppression,showing that these miRNAs are not ideal biomarkers for monitoring rejection of deep-buried nerve allografts, a response usuallyobserved later. Conclusions. We identified nine upregulated circulating miRNAs, whichmay have a biological function, particularlyduring the early stages after nerve allotransplantation under FK506 immunosuppression.

1. Background

At present, nerve allograft transplantation is a clinical realityfor select patients who sustain considerable nerve injury withnerve defect [1]. In the absence of host immunosuppres-sion, rejection of the nerve allograft typically occurs within7 d of transplantation [2]. An immunosuppressive agent,FK506 (tacrolimus), a macrolide antibiotic derived fromStreptomyces tsukubaensis, is used to suppress immunologicalresponses and demonstrates relatively good results withoutsignificant rejection when applied to nerve allografts [1].Following 3 d of pretreatment, FK506 immunosuppression

(1mg/(kg⋅d)) is recommended until the nerve growth hascrossed the distal graft site [3]. Importantly, this compoundenhances neuroregenerative effects; therefore, it shortens therequired duration of immunosuppression [4–6]. However,although adequate immunosuppression after nerve allotrans-plantation is important for successful nerve regenerationthrough the allograft, the detailed mechanism underlying therole of FK506 in the immunosuppression of the nerve allo-graft and promotion of nerve regeneration is poorly under-stood [7–9]. Lack of noninvasive biomarkers of rejectionmayalso preclude the optimization of therapeutic intervention.

Hindawi Publishing CorporationDisease MarkersVolume 2015, Article ID 863192, 9 pageshttp://dx.doi.org/10.1155/2015/863192

2 Disease Markers

Micro-RNAs (miRNAs) are endogenous, small (com-prising ∼22 nucleotides (nt)), single-stranded, and noncod-ing RNAs that regulate gene expression and are detectablein whole blood, serum, plasma, urine, and other body fluidsin a highly stable form [10–12]. Increasing evidence indicatesthat miRNAs play important roles in the posttranscriptionalregulation of gene expression and are involved in thecontrol of multiple biological and metabolic processes[13]. Circulating miRNAs present a potential technologicaladvantage because of their remarkable stability as well aspossible noninvasive and rapid diagnosis. Recently, RNAdeep sequencing using next-generation sequencing has beenshown to reveal a snapshot of RNA presence and quantityin a genome at a given moment in time [14]. The approachwith RNA-seq is highly suited for small RNA discovery,which not only provides sequences of low abundance speciesbut also provides quantitative data as the frequency ofsequencing reads reflects the abundance of miRNAs inthe population [15–17]. To date, little is known about theexpression and involvement of circulating miRNAs in nerveallograft transplantation. This study aimed to establish theexpression profile of circulating miRNAs with Illumina smallRNA deep sequencing during nerve allograft transplantationin the presence or absence of immunosuppression.

2. Materials and Methods

2.1. Experimental Design. Male BALB/c and C57BL/6 mice(age, 10–12 weeks; weight, 30–35 g) were purchased fromBioLasco (Yi-Lan, Taiwan). The BALB/c mice served assciatic nerve allograft donors. The C57BL/6 mice were ran-domized into two groups of nerve allograft transplantation:one with FK506 treatment and another without FK506treatment. These species of mice were selected on the basisof disparity at the MHC locus and prior experience withreciprocal rejection of grafts between these murine strains[18]. The mice were anesthetized by intraperitoneal injectionof an anesthetic cocktail consisting of 0.1mg/g ketamine and0.01mg/g xylazine. The anesthetized mice were restrainedin a supine position on a heated pad to maintain the bodytemperature at 37∘C. Under aseptic conditions, with sterilepovidone/iodine preparation and 70% ethanol and sterileinstruments and drapes, the skin over the proximal righthindlimb was incised, and the underlying biceps femorismuscle was bluntly dissected to expose the sciatic nerve. Anestablished mouse sciatic nerve allotransplantation modelwas used. In brief, 1 cm BALB/c donor sciatic nerve graftswere transplanted in reverse orientation into the 0.5-cmsciatic nerve gaps created in the recipient BALB/c mice.Tension-free repair was then performed under an operatingmicroscope with three 11–0 nylon (Ethicon Inc., Somerville,NJ) interrupted epineurial sutures, under 40x magnification.The muscle was closed with 5–0 vicryl sutures, and theskin was closed, with interrupted 5–0 nylon sutures. Theanimals were monitored to ensure appropriate feeding anddiet after surgery. FK506 (1mg/(kg⋅d)) was administeredsubcutaneously throughout the experimental course. At 3,7, and 14 d after initial surgery (𝑛 = 3 animals/group ateach time point), whole blood samples were collected at the

indicated times in tubes containing anticoagulant. The labels(F)3d, (F)7d, and (F)14d were used to indicate the serumsamples of mice undergoing allotransplantation with FK506immunosuppression for 3, 7, and 14 d, respectively, whilethe labels (N)3d, (N)7d, and (N)14d were used to indicatethe serum samples of mice undergoing allotransplantationwithout FK506 immunosuppression and sacrificed at post-operative days 3, 7, and 14, respectively. After the wholeblood samples were incubated at room temperature for15min, they were centrifuged at 3,000×g for 10min, whiteblood cells were slowly removed from the correspondinglayers, and the serum was extracted and stored at –80∘Cbefore processing for RNA analyses. All housing conditionsand surgical procedures, analgesia, and assessments werein accordance with national and institutional guidelines; anAssociation for Assessment and Accreditation of LaboratoryAnimal Care- (AAALAC-) accredited SPF facility was used.Animal protocols were approved by the Institutional AnimalCare and Use Committee (IACUC) of Kaohsiung ChangGung Memorial Hospital.

2.2. RNA Isolation. Total RNA was extracted from the har-vested nerve graft, whole blood, and serum by using themirVana miRNA Isolation Kit (Ambion, Austin, TX, USA).For miRNA array, purified RNA yield was determined onthe basis of the absorbance at 260 nm by using an SSP-3000 NanoDrop spectrophotometer (Infinigen Biotech, Cityof Industry, CA, USA), and RNA quality was evaluated witha Bioanalyzer 2100 system (Agilent Technologies, Palo Alto,CA, USA).

2.3. Small RNA Library Preparation. Small RNAs had linkersligated to them and bar-coded cDNAs were prepared usingthe TruSeq Small RNA Sample Prep Kit (Illumina) followingthe manufacturer’s instructions. Individual libraries wereanalyzed on a Bioanalyzer (Agilent) to detect the presenceof linkered cDNA of the appropriate size (135–165 bp), and11 bar-coded libraries were pooled into one sample by mixing2 ng of the 135–165 bp peak from each sample, as determinedby the Bioanalyzer.The 135–165-bp peak of the pooled cDNAswas purified from the mixed sample by using the Pippin PrepDNA Size Selection System (Sage Science) and confirmed bythe Bioanalyzer.

2.4. Illumina Small RNADeep Sequencing. Sequencing of thepooled libraries was performed in one lane of the IlluminaHiSeq2000 Sequencer at the miRBase v19, and 50-bp single-end reads of the libraries were obtained. After indexing andtrimming linker sequences, reads with a minimum lengthof 15 nt and with <3 terminal mismatches in the sequencewere sorted and counted for the following analysis. Thedeep sequencing experiment was performed by BGI TechSolutions Co., Ltd. (BGI Tech, Shenzhen, China).

2.5. Gene Ontology (GO) Enrichment and Kyoto Encyclopediaof Genes and Genomes (KEGG) Pathway Analyses. First, thismethod mapped all target gene candidates to GO termsin the database (http://www.geneontology.org/), calculated

Disease Markers 3

gene numbers for each term, and then used hypergeometrictest to find significantly enriched GO terms in target genecandidates, compared to the reference gene background.The Bonferroni correction for 𝑝 value was used to obtain acorrected 𝑝 value. GO terms with a corrected 𝑝 value of ≤0.05 were defined as significantly enriched in the target genecandidates. Pathway analysis from a major public pathway-related database KEGG [19] was performed to identify signif-icantly enriched metabolic pathways or signal transductionpathways involving the target gene candidates, compared tothe reference gene background. Genes with a false discoveryrate of ≤0.05 were considered as significantly enriched in thetarget gene candidates.

3. Results

3.1. Sequence Analysis of Small RNAs. To profile the circu-lating miRNAs expressed during nerve allograft transplan-tation, six small RNA libraries were generated from 3-, 7-,and 14-day serum samples of mice receiving nerve allografttransplantation with or without FK506 immunosuppression(hereafter indicated as (F)3d, (F)7d, (F)14d, (N)3d, (N)7d, and(N)14d).The libraries were sequenced by Illumina small RNAdeep sequencing technology. The number and proportionof the categories of small RNAs found are supplied inthe Supplementary File (in Supplementary Material avail-able online at http://dx.doi.org/10.1155/2015/863192). In total,7785957, 5978171, 5978171, 5574644, 5988924, and 5975945high-quality raw reads were obtained from the serumlibraries of (F)3d, (F)7d, (F)14d, (N)3d, (N)7d, and (N)14dmice, respectively. After filtering the low-quality sequences,empty adaptors, and single-read sequences, 5458187 (70.1%),5272112 (88.19%), 5670065 (94.85%), 5199001 (93.26%),5710818 (95.36%), and 5334192 (89.26%) clean reads, 18–30 nt in length, were selected for further analysis fromthe serum libraries of (F)3d, (F)7d, (F)14d, (N)3d, (N)7d,and (N)14d mice, respectively (Supplementary File 1: TableS1). Among the selected reads, 2689173, 2853061, 2750572,2615969, 3120416, and 2574471 sequences from the serumlibraries of (F)3d, (F)7d, (F)14d, (N)3d, (N)7d, and (N)14dmice, respectively, mapped perfectly to the mouse genome,amounting to 44.14% and 49.94% of the total reads, respec-tively; and 2602557, 2763676, 2657576, 2590795, 3088195,and 2555335 reads in the serum libraries of (F)3d, (F)7d,(F)14d, (N)3d, (N)7d, and (N)14d mice, respectively, werefound to be similar to miRNAs. The rest of the sequenceswere found to be other types of RNA, including noncodingRNA, rRNA, scRNA, snRNA, snoRNA, srpRNA, and tRNA.The size distribution of small RNAs (sRNAs) was similar inthe two libraries, and majority of them ranged from 21 to24 nt in length (Figure 1). The most abundant size class was22 nt, which accounted for 36.68%, 37.27%, 36.49%, 29.28%,34.98%, and 33.73% of the total reads in (F)3d, (F)7d, (F)14d,(N)3d, (N)7d, and (N)14dmice, respectively, followed by 23 nt(34.33%, 34.01%, 34.76%, 29.57%, 32.87%, and 37.67%, resp.).

3.2. Identification of Known miRNAs. The Illumina smallRNA deep sequencing approach allows us to determine therelative abundance of various miRNA families by calculating

the sequencing frequency. To investigate the expressionof known miRNAs during nerve allograft transplantation,identified small RNA sequences were compared with knownmature miRNAs in miRBase (version 19, 2012). The resultsshowed that 507 mature miRNAs were present in these sixlibraries (Supplementary File 1: Table S2). Among them,there were 147 mature miRNAs with ≥50 sequence reads(Supplementary File 1: Table S3) and nine mature miRNAswith ≥5000 sequence reads in at least one of these six libraries(Supplementary File 1: Table S4). In these libraries, knownmiRNAs had a broad range of expression levels; some (suchas miR-486-5p and miR-3107-5p) were found to have morethan hundreds of thousands of sequence reads, while manyothers had ≤20, indicating that expression varies significantlyamong different miRNA families.The proportion of differentcategories of small RNAs often reflects the roles in a particularstage and the associated biological mechanisms during nervegraft allotransplantation. With ≥5,000 reads, the followingfour miRNAs were dominantly expressed in all these sixlibraries: miR-25-3p, miR-486-5p, miR-3107-5p, and miR-92a-3p (Supplementary File 1: Table S5). In addition to miR-486-5p and miR-3107-5p, which have ≥200000 sequencereads in all six libraries, miR-92a-3p and miR-25-3p werethe third and fourth most abundant miRNA in (F)7d (22068and 13644 reads, resp.), (F)14d (19175 and 15427 reads, resp.),(N)3d (18312 and 10353 reads, resp.), and (N)14d (19852 and8768 reads, resp.) samples (Supplementary File 1: Table S6).

3.3. Differentially Expressed miRNAs. According to thechanges in relative miRNA abundance between the twoserum libraries obtained from mice receiving nerve allo-graft transplantation with and without FK506 immuno-suppression at the same indicated time points (3, 7, and14 d), unsupervised hierarchical clustering (Figure 2) as wellas supervised comparison of all significantly differentiallyexpressed miRNAs was conducted to categorize the samplesfrom experimental subjects at different days into differentgroups by using univariate analyses (𝑝 < 0.001). Figure 3illustrates the differentially expressed circulating miRNAsat different times between mice receiving allotransplan-tation with and without FK506 immunosuppression. Thedifferentially expressed miRNAs with ≥500 sequence readsin at least one of the libraries were selected for furthercomparison, which identified nine miRNAs with differencesgreater than 5-fold between the two libraries at 3 d (Table 1),but none was identified at 7 and 14 d, respectively. Amongthe nine upregulated miRNAs (let-7e-5p, miR-101a-3p, miR-151-5p, miR-181a-5p, miR-204-5p, miR-340-5p, miR-381-3p,miR-411-5p, miR-9-5p, and miR-219-2-3p), miR-9-5p had thehighest fold-change (≥50-fold at 3 d), followed by miR-340-5p with 38.8-fold. Therefore, we focused on the differentiallyexpressed miRNAs at 3 d with ≥500 sequence reads andstudied their expression profile throughout the course of theexperiment.Nine candidatemiRNAs (let-7e-5p,miR-101a-3p,miR-151-5p,miR-181a-5p,miR-204-5p,miR-340-5p,miR-381-3p, miR-411-5p, and miR-9-5p) were identified. Although allthese nine miRNAs had high-to-very-high sequence reads(576–9474) in the mice undergoing allotransplantation withFK506 immunosuppression at 3 d, compared to that observed

4 Disease Markers

Freq

uenc

y (%

) 40

30

20

10

014 16 18 20 22 24 26 28 30 32 34 36 38 40 42 4410 12

0.19

0.19

0.23

0.23

0.27

0.33

0.55

0.50

0.52

0.51

1.54

16.48

36.68

34.33

4.64

1.40

0.40

0.24

0.17

0.14

0.10

0.07

0.05

0.04

0.02

0.01

0.01

0.09

0.04

0.01

Length (nt)

(a)

Freq

uenc

y (%

) 40

30

20

10

014 16 18 20 22 24 26 28 30 32 34 36 38 40 42 4410 12

0.05

0.05

0.07

0.05

0.06

0.08

0.10

0.13

0.18

0.46

1.78

17.45

37.27

34.01

5.55

1.38

0.31

0.18

0.16

0.14

0.10

0.06

0.06

0.05

0.04

0.02

0.02

0.02

0.02

0.01

0.01

0.07

0.05

0.01

0.01

Length (nt)

(b)

Freq

uenc

y (%

)

40

30

20

10

0

14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 4410 12

0.21

0.17

0.19

0.17

0.21

0.26

0.51

0.35

0.42

0.47

1.48

15.10

36.49

34.76

5.23

1.63

0.45

0.29

0.24

0.23

0.17

0.15

0.15

0.12

0.10

0.07

0.06

0.04

0.04

0.03

0.02

0.11

0.06

0.01

0.01

Length (nt)

(c)

Freq

uenc

y (%

) 30

15

0

14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 4410 12

1.40

1.28

1.36

1.25

1.42

1.53

1.42

1.22

1.77

1.06

2.23

13.98

29.28

29.57

4.96

2.01

0.66

0.50

0.45

0.39

0.39

0.34

0.30

0.28

0.19

0.12

0.11

0.10

0.08

0.07

0.06

0.10

0.07

0.03

0.03

Length (nt)

(d)

Freq

uenc

y (%

)

40

30

20

10

014 16 18 20 22 24 26 28 30 32 34 36 38 40 42 4410 12

0.33

0.33

0.45

0.43

0.52

0.52

0.48

0.43

0.47

0.53

1.60

16.51

34.98

32.87

5.85

1.82

0.41

0.24

0.20

0.19

0.16

0.12

0.10

0.08

0.08

0.04

0.03

0.02

0.02

0.02

0.01

0.08

0.05

0.03

Length (nt)

(e)

Freq

uenc

y (%

) 40

30

20

10

014 16 18 20 22 24 26 28 30 32 34 36 38 40 42 4410 12

0.10

0.08

0.11

0.09

0.13

0.12

0.15

0.15

0.30

0.25

1.09

13.53

33.73

37.67

7.09

2.49

0.51

0.30

0.29

0.27

0.29

0.21

0.17

0.17

0.16

0.12

0.08

0.07

0.06

0.04

0.03

0.09

0.06

0.01

0.02

Length (nt)

(f)

Figure 1: Length distribution and abundance of small RNA sequences as observed by Illumina small RNA deep sequencing in six libraries.Nerve allograft transplantation with FK506 immunosuppression at 3 d (a), 7 d (b), or 14 d (c). Nerve allograft transplantation without FK506immunosuppression at 3 d (d), 7 d (e), or 14 d (f).

Table 1: Differentially expressedmiRNAs with ≥500 sequence readsand regulated ≥5-fold in the sera of C57BL/6 mice receiving nerveallotransplantation with or without immunosuppression for 3 d∗∗, 𝑝value < 0.01.

miR name Fold-change 𝑝 valuemmu-let-7e-5p 7.7 0

∗∗

mmu-miR-101a-3p 5.4 4.4𝐸 − 274

∗∗

mmu-miR-151-5p 5.8 4.1𝐸 − 150

∗∗

mmu-miR-181a-5p 5.2 0

∗∗

mmu-miR-204-5p 6.5 0

∗∗

mmu-miR-340-5p 38.8 8.3𝐸 − 32

∗∗

mmu-miR-381-3p 9.2 8.6𝐸 − 176

∗∗

mmu-miR-411-5p 7.0 4.7𝐸 − 186

∗∗

mmu-miR-9-5p >50 8.3𝐸 − 32

∗∗

in those without immunosuppression, none of thesemiRNAsshowed high expression at 7 d or 14 d (Figure 4).These resultsindicate that different miRNAs have clearly different specificexpression levels at different times.

3.4. Target Prediction and Functional Annotation. To furtherunderstand the physiological functions and biologicalprocesses involving the nine upregulatedmiRNAs at 3 d in thesera of mice receiving nerve allotransplantation with FK506

treatment, target prediction was performed by integratingfive common public databases: miRanda (http://www.mic-rorna/home.do), MirTarget2 (http://mirdb.org/miRDB/),PicTar (http://pictar.mdc-berlin.de/), PITA (http://genie.weizmann.ac.il/index.html), and RNAhybrid (http://bibis-erv.techfak.uni-bielefeld.de/rnahybrid/). Only the gene withmiRNA target interactions predicted by at least three of thesefive target prediction programs was chosen (SupplementaryFile 2). GO enrichment and KEGG pathway analyses wereperformed to identify the functional modules regulated bythese ninemiRNAs.The results ofGOannotation enrichmentanalysis regarding biological processes, cellular components,and molecular functions are listed in Supplementary File 3.KEGG pathway annotation showed that 21,703 backgroundgenes were annotated for 270 biological functions. KEGGpathway analysis revealed 39 pathways that were significantlyenriched with these miRNA targets (Supplementary File 4).Among them, the MAPK signaling pathway, Wnt signalingpathway, pathways involved in cancer, focal adhesion, gapjunction formation, and degradation, and axon guidance,calcium signaling pathway, and Notch signaling pathwaywere ranked among the most enriched pathways. Theseresults suggested that, during early stages, these targets showa high possibility of being regulated by miRNAs in cases ofnerve allotransplantation with FK506 immunosuppression,although the possibility of false-positive prediction from theprediction algorithm always exists.

Disease Markers 5

mmu-miR-19a-3pmmu-miR-142-3pmmu-miR-19b-3pmmu-miR-21a-5pmmu-miR-301a-3pmmu-miR-130b-5pmmu-miR-221-5p

mmu-miR-192-5pmmu-miR-148a-3pmmu-miR-222-5pmmu-miR-5113

mmu-miR-342-3pmmu-miR-130a-3pmmu-miR-148b-5pmmu-miR-145a-5pmmu-miR-222-3pmmu-miR-128-3pmmu-miR-24-3pmmu-miR-143-3pmmu-miR-126-5pmmu-miR-18a-5pmmu-miR-350-3pmmu-miR-674-5pmmu-miR-183-5pmmu-miR-1982.1-3pmmu-miR-1982.2-3pmmu-miR-16-2-3pmmu-miR-1249-3pmmu-miR-210-5pmmu-miR-200a-3pmmu-miR-223-5pmmu-miR-1943-5pmmu-miR-196a-5pmmu-miR-196b-5pmmu-miR-335-3pmmu-miR-3969mmu-miR-200b-3pmmu-miR-1843a-3pmmu-miR-582-3pmmu-miR-125b-2-3pmmu-miR-872-5pmmu-miR-99a-3pmmu-miR-499-5pmmu-miR-214-5pmmu-miR-3068-3pmmu-miR-16-1-3pmmu-miR-92a-1-5pmmu-miR-322-5pmmu-miR-301b-3pmmu-miR-615-3pmmu-miR-193b-3pmmu-miR-345-5pmmu-miR-133b-3pmmu-miR-429-3pmmu-miR-423-3pmmu-miR-181a-2-3pmmu-miR-18a-3pmmu-miR-486-3pmmu-miR-3107-3pmmu-miR-92a-3pmmu-miR-328-3pmmu-miR-1198-5pmmu-miR-484mmu-miR-375-3pmmu-miR-106b-3pmmu-miR-130b-3pmmu-miR-3074-5pmmu-miR-339-3pmmu-miR-142-5pmmu-miR-223-3pmmu-miR-199a-3pmmu-miR-199b-3pmmu-miR-23a-3pmmu-miR-28a-3pmmu-miR-350-5pmmu-miR-17-5pmmu-miR-10a-5pmmu-miR-10b-5pmmu-miR-425-5pmmu-miR-210-3pmmu-let-7d-3pmmu-miR-221-3pmmu-miR-151-3pmmu-miR-532-5pmmu-miR-16-5pmmu-miR-25-3pmmu-miR-93-5pmmu-miR-451ammu-miR-30e-5pmmu-miR-215-3pmmu-miR-106b-5pmmu-miR-101b-3pmmu-miR-215-5pmmu-miR-664-5pmmu-miR-676-5pmmu-miR-3473dmmu-miR-324-5pmmu-miR-872-3pmmu-miR-140-5pmmu-miR-186-5pmmu-miR-30a-5pmmu-miR-30c-5pmmu-miR-30d-5pmmu-miR-181d-5pmmu-miR-1839-5pmmu-miR-15a-5pmmu-miR-182-5pmmu-miR-421-3pmmu-miR-203-3pmmu-miR-30b-5pmmu-miR-126-3pmmu-miR-29a-3pmmu-miR-532-3pmmu-miR-199a-5pmmu-miR-351-5pmmu-miR-540-3pmmu-miR-133a-3pmmu-miR-298-5pmmu-miR-146a-5pmmu-miR-574-3pmmu-miR-676-3pmmu-miR-191-5pmmu-miR-378a-3pmmu-miR-140-3pmmu-miR-148a-5pmmu-miR-22-3pmmu-miR-1843b-5pmmu-miR-1843a-5pmmu-miR-365-3pmmu-miR-668-3pmmu-miR-214-3pmmu-miR-26b-3pmmu-miR-669p-5pmmu-miR-669a-5pmmu-miR-24-2-5pmmu-miR-322-3pmmu-miR-345-3pmmu-miR-326-3pmmu-miR-501-3pmmu-miR-296-5pmmu-miR-150-5pmmu-miR-378cmmu-miR-107-3pmmu-miR-27a-3pmmu-miR-483-3pmmu-miR-1954mmu-miR-150-3pmmu-miR-144-3pmmu-miR-17-3pmmu-miR-141-3pmmu-miR-15b-3pmmu-miR-674-3pmmu-miR-21a-3pmmu-miR-3102-3p.2-3pmmu-miR-3072-3pmmu-miR-378dmmu-miR-455-5pmmu-miR-194-2-3pmmu-miR-122-3pmmu-miR-5105mmu-miR-7a-2-3pmmu-miR-3096a-5pmmu-miR-467d-5pmmu-miR-3470bmmu-miR-3058-3pmmu-miR-652-3pmmu-miR-93-3pmmu-miR-425-3pmmu-miR-122-5pmmu-miR-434-3pmmu-miR-205-5pmmu-miR-505-5pmmu-miR-802-5pmmu-miR-339-5pmmu-miR-486-5pmmu-miR-3107-5pmmu-miR-702-3pmmu-miR-1843b-3pmmu-miR-3473bmmu-miR-1964-3pmmu-miR-3473emmu-miR-1196-5pmmu-miR-1306-3pmmu-miR-15a-3pmmu-let-7f-5pmmu-miR-98-5pmmu-miR-15b-5pmmu-miR-25-5pmmu-miR-6243mmu-miR-132-3pmmu-miR-5109mmu-miR-490-3pmmu-miR-320-3pmmu-miR-744-5pmmu-miR-423-5pmmu-miR-30c-2-3pmmu-miR-128-1-5p

mmu-miR-673-5pmmu-miR-1947-5pmmu-miR-5107-3pmmu-miR-1981-5pmmu-miR-219-1-3pmmu-miR-361-3pmmu-miR-31-5pmmu-miR-361-5pmmu-miR-99b-3pmmu-miR-700-5pmmu-miR-195a-3pmmu-miR-20a-5pmmu-miR-28a-5pmmu-miR-125b-1-3pmmu-miR-99a-5pmmu-miR-100-5pmmu-miR-877-5pmmu-miR-23b-3pmmu-miR-125b-5pmmu-miR-30e-3pmmu-miR-146b-5pmmu-miR-342-5pmmu-miR-181c-5pmmu-miR-27b-3pmmu-miR-181c-3pmmu-miR-497-5pmmu-miR-26a-5pmmu-miR-151-5pmmu-miR-181b-5pmmu-miR-181a-5pmmu-miR-181a-1-3pmmu-miR-410-3pmmu-miR-139-5pmmu-miR-34c-5pmmu-miR-103-3pmmu-miR-340-5pmmu-miR-101a-3pmmu-miR-136-3pmmu-miR-204-5p

mmu-miR-411-5p

mmu-miR-3082-3pmmu-miR-194-5pmmu-miR-374b-5pmmu-miR-138-5pmmu-miR-671-3pmmu-miR-324-3pmmu-miR-1948-3pmmu-miR-30c-1-3p

mmu-miR-99b-5pmmu-miR-125a-5pmmu-miR-149-5pmmu-miR-127-3pmmu-miR-92b-3pmmu-miR-3470ammu-miR-433-3pmmu-miR-134-5pmmu-miR-299a-5pmmu-miR-29c-3pmmu-miR-431-5pmmu-miR-195a-5pmmu-miR-381-3pmmu-miR-300-3pmmu-miR-1298-5pmmu-miR-335-5pmmu-miR-124-5pmmu-miR-504-5pmmu-miR-323-5pmmu-miR-370-3pmmu-miR-874-5pmmu-miR-708-3pmmu-miR-7a-1-3pmmu-miR-136-5pmmu-miR-409-3pmmu-miR-129-2-3pmmu-miR-485-3p

mmu-miR-330-3pmmu-miR-363-3pmmu-miR-29b-3pmmu-miR-127-5pmmu-miR-592-5pmmu-miR-485-5pmmu-miR-129-5pmmu-miR-874-3pmmu-miR-218-5pmmu-miR-138-1-3pmmu-miR-129-1-3pmmu-miR-338-5pmmu-miR-153-3pmmu-miR-9-3pmmu-miR-431-3pmmu-miR-487b-3pmmu-miR-323-3pmmu-miR-654-3pmmu-miR-124-3pmmu-miR-219-2-3pmmu-miR-338-3pmmu-miR-9-5pmmu-miR-3096b-5pmmu-let-7i-5p

mmu-let-7d-5pmmu-miR-3057-5p

mmu-miR-1968-5pmmu-miR-144-5p

mmu-miR-330-5pmmu-miR-148b-3pmmu-miR-26b-5pmmu-miR-30a-3pmmu-miR-1306-5pmmu-miR-672-5pmmu-miR-1970mmu-miR-511-5pmmu-miR-434-5p

mmu-miR-1934-5pmmu-miR-344d-3p

mmu-miR-27b-5pmmu-miR-145a-3p

mmu-miR-1945mmu-miR-184-3pmmu-miR-5107-5pmmu-miR-541-5pmmu-miR-155-5pmmu-miR-664-3pmmu-miR-666-5pmmu-miR-1960

mmu-miR-30d-3p

mmu-miR-5128

mmu-let-7e-5p

mmu-let-7b-3p

mmu-let-7b-5p

mmu-let-7a-5p

mmu-let-7g-5p

mmu-let-7c-5p

5 4 3 2 13

(d)7 14

−4 0 +4

Gray: missing data

log2(treatment/control)

Figure 2: Unsupervised hierarchical clustering of miRNA expression. Hierarchical clustering of miRNA differentially expressed in the seraof the mice receiving nerve allotransplantation with or without daily injection of 1mg/(kg⋅d) FK506 for immunosuppression.

6 Disease Markers

Expression level

Expr

essio

n le

vel

106

106

00

Upexpressed miRNADownexpressed miRNAEqually expressed miRNA

FK5063

d

Control 3d

(a)

Expr

essio

n le

vel

106

0

Expression level

1060


FK5067

dControl 7d

(b)

Expr

essio

n le

vel

106

0

Expression level

1060


FK50614

d

Control 14d

(c)

Figure 3: Comparison of miRNA expression levels at 3, 7, and 14 d in the sera of the mice receiving nerve allotransplantation with or withoutdaily injection of 1mg/(kg⋅d) FK506 for immunosuppression. Red dots: upregulated miRNAs, green dots: downregulated miRNAs, and bluedots: equally expressed miRNAs.

Disease Markers 7Se

quen

ce re

ads

Sequ

ence

read

s

100008000600040002000

200

150

100

50

03 7 14 3 7 14 3 7 14

3 7 14 3 7 14 3 7 14

3 7 14 3 7 14 3 7 14

mmu-let-7e-5p mmu-miR-101a-3p2000

1500

1000

5005004003002001000

2000

1500

1000

5004003002001000

2000

1500

1000

5005004003002001000

2000

1500

1000

5005004003002001000

Sequ

ence

read

sSe

quen

ce re

ads

Sequ

ence

read

s

Sequ

ence

read

sSe

quen

ce re

ads

Sequ

ence

read

s

Sequ

ence

read

s

2000

1500

1000

5005004003002001000

+FK506−FK506

+FK506−FK506

+FK506−FK506

mmu-miR-151-5p

mmu-miR-181a-5p mmu-miR-204-5p mmu-miR-340-5p

mmu-miR-381-3p mmu-miR-411-5p mmu-miR-9-5p

100000

90000

80000

700006000020000

15000

1000

5000

0

2000180016001400120010005004003002001000

1000090008000700060005000200

150

100

50

0

(d) (d) (d)

(d) (d) (d)

(d) (d) (d)

Figure 4: Sequence reads of the nine upregulated circulating miRNAs throughout the experimental period in the sera of the mice receivingnerve allotransplantation with or without daily injection of 1mg/(kg⋅d) FK506 for immunosuppression.

4. Discussion

The Illumina deep sequencing platform is efficient formiRNA discovery and is widely used to generate small RNAprofiles in various organisms. In this study, we identifiedthe circulating miRNAs in a mouse model of nerve allo-graft transplantation under FK506 immunosuppression byIllumina small RNA deep sequencing. The sequence analysisshowed that the dominant size of small RNAs in circulationafter nerve allotransplantation in the presence or absenceof FK506 immunosuppression was 22 nt, followed by 23 nt.This result is typical of Dicer-processed small RNAs andwas consistent with the known size of ∼22 nt for miRNAs.Selection of the differentially expressed miRNAs under therelatively strict conditions (≥500 sequence reads in at leastone of the libraries selected for comparison, ≥5-fold dif-ference in expression, and a 𝑝 value of ≤ 0.01) identifiednine upregulated miRNAs (let-7e-5p, miR-101a-3p, miR-151-5p, miR-181a-5p, miR-204-5p, miR-340-5p, miR-381-3p, miR-411-5p, miR-9-5p, and miR-219-2-3p) at 3 d, but none at 7 d

or 14 d, suggesting that these upregulated miRNAs impactbiological functions, particularly during the early stages afternerve allotransplantation with FK506 immunosuppression.

Among these nine upregulated circulatingmiRNAs,miR-9-5p had the highest fold-change of ≥50-fold, followed bymiR-340-5p with 38.8-fold. Correlation of miR-9-3p withthe genes involved in immune/inflammatory responses (e.g.,IFNG and IL17F), apoptosis (e.g., PDCD4 and PTEN), andcell proliferation (e.g., NKX3-1 and GADD45A) has beenreported in stimulated human peripheral blood lymphocytes[20]. miR-340-5p is confirmed to be related to nerve injuryand elevated at all five time points, from 1 h to 7 d, aftertraumatic brain injury [21]. Further, none of the remainingseven upregulated miRNAs has been explored or validated,in available literature, to be related to immune function,nerve injury, or nerve regeneration. In this study, KEGGpathway analysis of these miRNA targets revealed thatMAPK signaling pathway, Wnt signaling pathway, pathwaysinvolved in cancer, focal adhesion, gap junction formationand degradation, and axon guidance, calcium signaling

8 Disease Markers

pathway, and Notch signaling pathway are ranked among themost enriched pathways. Although most of these pathwayshave been reported to be involved in the processes duringnerve injury or nerve regeneration, these pathways are quiteheterogeneous, and their role in nerve allotransplantation hasseldombeen explored.However, it is important to identify theorigin and target cells of these circulating miRNAs prior tothe discussion of these pathways. Whether these circulatingmiRNAshave originated fromTcells, blood cells, or endothe-lial cells of the circulating system is unknown, and their targetcells are yet to be identified. Unfortunately, at present, thereis no standard protocol to identify the origin of circulatingmiRNAs. Notably, although the origin of circulatingmiRNAsis debatable, the expression profile of circulating miRNAs isobviously different from that of the miRNAs in pathologicaltissues [11, 22].The immunosuppressive drug FK506 has beenproved to exert neuroprotective and neurotrophic actionsin experimental models; it increases neurite elongation andaccelerates the rate of nerve regeneration in vitro and in vivo[7, 23]. In this study, miRNAs are differentially expressedbetween mice receiving allogeneic grafts with and withoutimmunosuppression during the early stages, suggesting thattheir important regulatory function was not the suppressionof graft rejection, a phenomenon that generally occurs at 7 dafter nerve allotransplantation in the absence of immunosup-pression. Whether these circulating miRNAs play a potentialrole in the nerve regeneration-enhancing activity of FK506requires further investigation.

Although allograft biopsy is the gold standard for diagno-sis of conditions such as acute rejection, disease recurrence,and drug toxicity [24, 25], it is impractical to acquire a nervebiopsy in clinical settings. Circulating miRNAs, owing to thenoninvasive nature of their detection, their disease specificity,and the availability of accurate techniques for detection andmonitoring, may be developed as excellent biomarkers ofallograft injury and function [26]. For example, it had beenreported that circulating cell-freemiRNAs correlate with spe-cific hepatic injury and thus may serve as feasible monitoringand outcome-predictive biomarkers in liver transplantation[27]. Differential expression of miRNAs was also observed inurine samples between patient groups with chronic allograftdysfunction [28]. Therefore, miRNAs may serve as potentialbiomarkers for organ quality, ischemia-reperfusion injury,acute rejection, tolerance, and chronic allograft dysfunction,emphasizing their mechanistic and clinical applications [26,29]. In this study, our additional goal was to identify possibleupregulated circulating miRNAs after nerve allotransplanta-tion in mice with or without immunosuppression and thendetermine whether these identified circulating miRNAs maybe used as targets for monitoring or therapeutic intervention.However, when compared to themiRNAs found inmice withuntreated isograft, no circulating miRNA with significantlyincreased expression was found at 7 d or 14 d in both groups.The presence of these nine upregulated circulating miRNAsonly at 3 d, but not at 7 d or 14 d, after nerve allotransplan-tation with FK506 immunosuppression, indicates that theseupregulatedmiRNAs are not ideal biomarkers formonitoringthe status of deep-buried nerve allografts. However, theunderlyingmechanistic basis for the expression of circulating

miRNA during the early stages of nerve allotransplantationunder immunosuppression, but not under allogeneic condi-tion, remains an area of utmost interest. In our prior reportthat profiles the circulating miRNAs expression in a mousemodel of nerve allotransplantation, we used BALB/c mice asrecipient animals and C57BL/6mice as donor animals for fullmajor histocompatibility complex (MHC) disparity [30, 31]and identified the circulating miR-320, miR-762, and miR-423-5p as potential biomarkers for monitoring the immuno-suppression status of the nerve allograft [18]. We also foundthat the expression of all these 3 upregulated circulatingmiRNAs significantly decreased at 2, 4, and 6 d after discon-tinuation of FK506 immunosuppression [18]. In contrast, inthis study that used C57BL/6 mice as recipient animals andBALB/cmice as donor animals, no ideal upregulatedmiRNAsare identified for monitoring the status of deep-buried nerveallografts. The results indicate that a distinct expression ofcirculating miRNAs may exist even between different kindsof the same species and also suggest the difficulty to translatethe research results of mice to the human being.

In conclusion, this study identified nine upregulated cir-culating miRNAs after nerve allotransplantation with FK506immunosuppression by Illumina small RNA deep sequenc-ing. These miRNAs affect biological functions, in particularduring the early stages after nerve allotransplantation underimmunosuppression.

Conflict of Interests

The authors have no personal financial or institutional inter-ests in any of the drugs, materials, or devices described in thispaper.

Authors’ Contribution

Ching-Hua Hsieh and Cheng-Shyuan Rau were responsiblefor the design and coordination of data acquisition andanalysis and paper writing. Shao-Chun Wu was responsiblefor the revision of the paper. Johnson Chia-Shen Yang partic-ipated by providing and coordinating the resources. Yi-ChunChen and Chia-Jung Wu were involved in the acquisitionof deep sequencing data. Tsu-Hsiang Lu, Yi-Chan Wu, Siou-Ling Tzeng, and Chia-Wei Lin contributed to animal surgeryand acquisition of study specimens. All authors read andapproved the final paper. Shao-ChunWu and Cheng-ShyuanRau indicated equal contribution in authorship.

Acknowledgments

The work was supported by Chang Gung Memorial Hospital(CMRPG8A0022 and CMRPG8A0023), Taiwan.The authorsthank the Genomic & Proteomic Core Laboratory, Depart-ment ofMedical Research, KaohsiungChangGungMemorialHospital, for supporting their analysis work.

References

[1] S. E. Mackinnon, V. B. Doolabh, C. B. Novak, and E. P. Trulock,“Clinical outcome following nerve allograft transplantation,”

Disease Markers 9

Plastic and Reconstructive Surgery, vol. 107, no. 6, pp. 1419–1429,2001.

[2] S. K. Sen, J. B. Lowe III, M. J. Brenner, D. A. Hunter, and S.E. Mackinnon, “Assessment of the immune response to dose ofnerve allografts,” Plastic and Reconstructive Surgery, vol. 115, no.3, pp. 823–830, 2005.

[3] W. Z. Ray and S. E. Mackinnon, “Management of nervegaps: autografts, allografts, nerve transfers, and end-to-sideneurorrhaphy,” Experimental Neurology, vol. 223, no. 1, pp. 77–85, 2010.

[4] N. Weinzweig, S. Grindel, M. Gonzalez, D. Kuy, J. Fang, and B.Shahani, “Peripheral-nerve allotransplantation in rats immuno-suppressed with transient or long-term FK-506,” Journal ofReconstructive Microsurgery, vol. 12, no. 7, pp. 451–459, 1996.

[5] M. J. Brenner, S. E. Mackinnon, S. R. Rickman et al., “FK506and anti-CD40 ligand in peripheral nerve allotransplantation,”RestorativeNeurology andNeuroscience, vol. 23, no. 3-4, pp. 237–249, 2005.

[6] J. R. Bain, “Peripheral nerve and neuromuscular allotransplan-tation: current status,”Microsurgery, vol. 20, no. 8, pp. 384–388,2000.

[7] P. Konofaos and J. K. Terzis, “FK506 and nerve regeneration:past, present, and future,” Journal of Reconstructive Micro-surgery, vol. 29, no. 3, pp. 141–148, 2013.

[8] I. Sosa, O. Reyes, and D. P. Kuffler, “Immunosuppressants:neuroprotection and promoting neurological recovery follow-ing peripheral nerve and spinal cord lesions,” ExperimentalNeurology, vol. 195, no. 1, pp. 7–15, 2005.

[9] E. C. Toll, A. M. Seifalian, and M. A. Birchall, “The roleof immunophilin ligands in nerve regeneration,” RegenerativeMedicine, vol. 6, no. 5, pp. 635–652, 2011.

[10] A. V. Vlassov, S. Magdaleno, R. Setterquist, and R. Conrad,“Exosomes: current knowledge of their composition, biologicalfunctions, and diagnostic and therapeutic potentials,” Biochim-ica et Biophysica Acta, vol. 1820, no. 7, pp. 940–948, 2012.

[11] C.-H. Hsieh, C.-S. Rau, J. C. Jeng et al., “Whole blood-derivedmicroRNA signatures in mice exposed to lipopolysaccharides,”Journal of Biomedical Science, vol. 19, article 69, 2012.

[12] C.-H. Hsieh, J. C.-S. Yang, J. C. Jeng et al., “CirculatingmicroRNA signatures in mice exposed to lipoteichoic acid,”Journal of Biomedical Science, vol. 20, article 2, 2013.

[13] V. Kinet, J. Halkein, E. Dirkx, and L. J. DeWindt, “Cardiovascu-lar extracellular microRNAs: emerging diagnostic markers andmechanisms of cell-to-cell RNA communication,” Frontiers inGenetics, vol. 4, article 214, 2013.

[14] Y. Chu and D. R. Corey, “RNA sequencing: platform selection,experimental design, and data interpretation,” Nucleic AcidTherapeutics, vol. 22, no. 4, pp. 271–274, 2012.

[15] J. Burnside, M. Ouyang, A. Anderson et al., “Deep sequencingof chickenmicroRNAs,”BMCGenomics, vol. 9, article 185, 2008.

[16] J. M. Dhahbi, S. R. Spindler, H. Atamna et al., “Deep sequencingidentifies circulating mouse miRNAs that are functionallyimplicated in manifestations of aging and responsive to calorierestriction,” Aging, vol. 5, no. 2, pp. 130–141, 2013.

[17] Q. Yang, J. Lu, S. Wang, H. Li, Q. Ge, and Z. Lu, “Applicationof next-generation sequencing technology to profile the circu-lating microRNAs in the serum of preeclampsia versus normalpregnant women,” Clinica Chimica Acta, vol. 412, no. 23-24, pp.2167–2173, 2011.

[18] C.-S. Rau, J. C.-S. Yang, S.-C. Wu et al., “Profiling circulatingmicroRNA expression in amousemodel of nerve allotransplan-tation,” Journal of Biomedical Science, vol. 20, article 64, 2013.

[19] M. Kanehisa, M. Araki, S. Goto et al., “KEGG for linkinggenomes to life and the environment,” Nucleic Acids Research,vol. 36, no. 1, pp. D480–D484, 2008.

[20] C. Girardi, C. De Pitta, S. Casara et al., “Integration analysis ofMicroRNA andmRNA expression profiles in human peripheralblood lymphocytes cultured in modeled microgravity,” BioMedResearch International, vol. 2014, Article ID 296747, 16 pages,2014.

[21] L. Liu, T. Sun, Z. Liu et al., “Traumatic brain injury dysregulatesMicroRNAs to modulate cell signaling in rat hippocampus,”PLoS ONE, vol. 9, no. 8, Article ID e103948, 2014.

[22] C.-H. Hsieh, J. C.-S. Yang, J. C. Jeng et al., “CirculatingmicroRNA signatures in mice exposed to lipoteichoic acid,”Journal of Biomedical Science, vol. 20, no. 1, article 2, 2013.

[23] R. Fedakar, A. H. Aydiner, and I. Ercan, “A comparison of‘Life threatening injury’ concept in the Turkish Penal Code andtrauma scoring systems,”Ulusal Travma ve Acil Cerrahi Dergisi,vol. 13, no. 3, pp. 192–198, 2007.

[24] W. W. Williams, D. Taheri, N. Tolkoff-Rubin, and R. B. Colvin,“Clinical role of the renal transplant biopsy,” Nature ReviewsNephrology, vol. 8, no. 2, pp. 110–121, 2012.

[25] S. G. Hubscher, “Transplantation pathology,” Seminars in LiverDisease, vol. 29, no. 01, pp. 74–90, 2009.

[26] V. R. Mas, C. I. Dumur, M. J. Scian, R. C. Gehrau, and D. G.Maluf, “MicroRNAs as biomarkers in solid organ transplanta-tion,” American Journal of Transplantation, vol. 13, no. 1, pp. 11–19, 2013.

[27] R. C. Gehrau, V. R. Mas, and D. G. Maluf, “Hepatic diseasebiomarkers and liver transplantation: what is the potentialutility of microRNAs?” Expert Review of Gastroenterology andHepatology, vol. 7, no. 2, pp. 157–170, 2013.

[28] M. J. Scian, D. G. Maluf, K. G. David et al., “MicroRNAprofiles in allograft tissues and paired urines associate withchronic allograft dysfunction with IF/TA,” American Journal ofTransplantation, vol. 11, no. 10, pp. 2110–2122, 2011.

[29] J. C. Spiegel, J. M. Lorenzen, and T.Thum, “Role of microRNAsin immunity and organ transplantation,” Expert Reviews inMolecular Medicine, vol. 13, article e37, 2011.

[30] T. H. H. Tung, S. E. Mackinnon, and T. Mohanakumar, “Long-term limb allograft survival using anti-CD401, antibody in amurine model,” Transplantation, vol. 75, no. 5, pp. 644–650,2003.

[31] J. N. Jensen, T. H. H. Tung, S. E. Mackinnon, M. J. Brenner, andD. A. Hunter, “Use of anti-CD40 ligand monoclonal antibodyas antirejection therapy in a murine peripheral nerve allograftmodel,”Microsurgery, vol. 24, no. 4, pp. 309–315, 2004.

Submit your manuscripts athttp://www.hindawi.com

Stem CellsInternational

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation http://www.hindawi.com Volume 2014

Immunology ResearchHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinson’s Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com

research article identification of circulating...

Documents