Research Article Identification of Aberrantly Expressed miRNAs in …downloads.hindawi.com/journals/grp/2014/473817.pdf · 2019. 7. 31. · Research Article Identification of Aberrantly

Research ArticleIdentification of Aberrantly Expressed miRNAs inGastric Cancer

Dan Liu Xiaowei Hu Hongfeng Zhou Guangyue Shi and Jin Wu

The Seventh Department of Internal Medicine The Affiliated Tumor Hospital of Harbin Medical UniversityHarbin Heilongjiang 150081 China

Correspondence should be addressed to Jin Wu medmst126com

Received 18 December 2013 Revised 31 March 2014 Accepted 1 April 2014 Published 1 June 2014

Academic Editor Sergio Morini

Copyright copy 2014 Dan Liu et alThis is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The noncoding components of the genome including miRNA can contribute to pathogenesis of gastric cancer Their expressionhas been profiled in many human cancers but there are a few published studies in gastric cancer It is necessary to identifynovel aberrantly expressed miRNAs in gastric cancer In this study the expression profile of 1891 miRNAs was analyzed using amiRCURY array LNA miRNA chip from three gastric cancer tissues and three normal tissues The expression levels of 4 miRNAswere compared by real-time PCR between cancerous and normal tissues We found that 31 miRNAs are upregulated in gastriccancer (119875 lt 005) and 10 miRNAs have never been reported by other studies 30 miRNA are downregulated (119875 lt 005) in gastriccancer tissues Gene ontology analysis revealed that those dysregulated miRNAs mainly take part in regulating cell proliferationThe levels of has-miR-105 -213lowast -514b and -548n were tested by real-time PCR and have high levels in cancerous tissues Here wereport a miRNA profile of gastric cancer and provide new perspective to understand this malignant diseaseThis novel informationsuggests the potential roles of these miRNAs in the diagnosis prognosis biomarkers or therapy targets of gastric cancer

1 Introduction

Gastric cancer is one of the most frequent cancers and isthe second leading cause of cancer mortality worldwide [1]Nearly half of gastric cancers occur in China most of whichare diagnosed when the disease has progressed to late stagesbecause of the nonspecific symptoms present at early stages[2] As a result the overall 5-year survival rate of gastriccancer was approximately 20 [3] Tumor markers have thepotential to improve the situation by screening high riskgroup at early stage Unfortunately the tumor markers suchas carcinoembryonic antigen (CEA) carbohydrate antigen19-9 (CA19-9) CA12-5 and CA72-4 have limitation perfor-mance in detecting gastric cancer because of low sensitivityand specificity [4] Thus there is a need to discover somenovel diagnostic biomarkers to allow early detection of thismalignancy It is known that several environmental factorsincluding diet high in salted and nitrated food tobacco usealcoholic consumptions and especially Helicobacter pylori(HP) infection [5] However the molecular pathogenesis ofgastric cancer still remains to be explored Mechanisms for

tumorigenesis and progression of gastric cancer have notyet been discovered and specific therapeutic targets have notbeen identified [6]

Micro-RNAs (miRNAs) are small noncoding regulatoryRNAs of about 19ndash22 nucleotides which function to bindthe 31015840 untranslated region of their target mRNAs resultingin translational inhibition or mRNA degradation [7] Whilethe biological roles ofmiRNAare under intense investigationthey are considered to control a variety of tumor cell func-tions including cell proliferation migration invasion anddifferentiation [8] A growing number of evidences suggestthe correlation altered miRNA expression and cancers Theaberrant expression of miRNAs referred to several importantprocesses during carcinogenesis Let-7 is one of the earliestidentifiedmiRNAs is significantly reduced in a large numberof malignancies and can attenuate the development of lungcancer [9] High-throughput techniques such as gene chiphave identified thousands of upregulations such as miR-21miR-17 and miR-92a whereas others tended to downregula-tion such as miR205 and miR-145 in cancerous tissues [10]Several studies have investigated the aberrantly expressed

Hindawi Publishing CorporationGastroenterology Research and PracticeVolume 2014 Article ID 473817 9 pageshttpdxdoiorg1011552014473817

2 Gastroenterology Research and Practice

miRNAs in gastric cancer miR-199a-3p is significantly higherin serum of gastric cancer patients and may be used asdiagnostic biomarker [4] MiR-34a was downregulated ingastric cancer cell lines [11 12] MiR-429 suppresses tumorcells proliferation and may serve as tumor suppressor duringtumorigenesis of gastric cancer [13] MiR-19ab regulatesmultidrug resistance in gastric cancer by targeting PTEN [14]Li et al had identified abnormal expressed miRNA profileswith 40 upregulatedmiRNAs and 36 downregulatedmiRNAsin intestinal-type gastric caners by miRNA array [6] It hasbeen estimated that there are about 1000 miRNAs in humanbut previous studies have only screened miRNA expressionin gastric cancer patients from less than 500 miRNAs It isnecessary to screen gastric cancer with larger collections ofmiRNAs by gene chip

To explore more related miRNAs we have used 6thgeneration of miRNA array that contains more than 1891probes which include nearly all human miRNAs and identi-fied new aberrant expressionmiRNAs between gastric cancerand normal tissues We identified 61 new obviously changedexpression miRNAs in gastric cancers Our data offer newclues to study the miRNA profiles that refer to molecularmechanism of gastric cancer carcinogenesis and providenovel biomarkers repertoire of this malignant disease

2 Material and Methods

The tissue samples in this study were derived from patientsundergoing a surgical procedure to remove a portion ofgastric cancer at the Affiliated Tumor Hospital of HarbinMedical University The collection of samples conformed tothe policies of China and practices of the facilityrsquos Institu-tional Review Board Upon removal of the surgical specimenresearch personnel immediately transported the tissue to thesurgical pathology laboratory Pathology faculty performeda gross analysis of the specimen and selected cancerousappearing gastric tissue and normal appearing gastric cancerfor research Each sample was placed in a cryovial andpreserved in minus80∘C refrigerator until analysis Subsequentlythe surgical specimens confirmed the histopathology of thesamples taken for research

21 Total RNA Isolation and Quality Analysis Frozen tissueswere first pulverized in a stainless steel mortar and pestleTotal RNA was isolated using TRIzol (Invitrogen CarslbadCA) and miRNeasy mini kit (QIAGEN) according to man-ufacturerrsquos instructions RNA quality and quantity weremeasured by using nanodrop spectrophotometer (ND-1000Nanodrop Technologies) and RNA integrity was determinedby gel electrophoresis

22 miRNA Precursor Expression Profiling After RNA iso-lation from the samples the miRCURY Hy3Hy5 powerlabeling kit (Exiqon Vedbaek Denmark) was used accordingto the manufacturerrsquos guideline for miRNA labelling Onemicrogram of each sample was 31015840-end-labeled with Hy3fluorescent label using T4 RNA ligase by the followingprocedure RNA in 20 120583L of water was combined with 10 120583L

of CIP buffer and CIP (Exiqon) The mixture was incubatedfor 30min at 37∘C and was terminated by incubation for5min at 95∘C Then 30 120583L of labeling buffer 15 120583L offluorescent label (Hy3) 20120583L of DMSO and 20 120583L oflabeling enzyme were added into the mixture The labelingreaction was incubated for 1 h at 16∘C and terminated byincubation for 15min at 65∘C After stopping the labelingprocedure the Hy3-labeled samples were hybridized onthe miRCURY LNA array (v160) (Exiqon) according toarray manual The total 25 120583L mixture from Hy3-labeledsamples with 25 120583L hybridization buffer was first denaturedfor 2min at 95∘C incubated on ice for 2min and thenhybridized to the microarray for 16ndash20 h at 56∘C in a 12-Bay Hybridization Systems (Hybridization System Nimble-gen Systems Inc Madison WI USA) which provides anactive mixing action and constant incubation temperatureto improve hybridization uniformity and enhance signalFollowing hybridization the slides were achieved washedseveral times usingWash buffer kit (Exiqon) and finally driedby centrifugation for 5min at 400 rpm Then the slides werescanned using the Axon GenePix 4000B microarray scanner(Axon Instruments Foster City CA)

23 miRNA Quantification by Real-Time RT-PCR (qRT-PCR)SYBRGreen RT-qPCR assay was used for miRNA quantifica-tion In brief one microgram of extracted RNA was reverse-transcribed per the manufacturerrsquos instructions qRT-PCRwas carried out in thin-wall PCR plates (Applied BiosystemsFoster City CA USA) Each reaction mixture contained125 120583L of SYBR Premix Ex Taq (2times) 05 lL of referencedye (ROX) II (50times) (Takara Otsu Japan) 05 120583L of forwardprimer (10120583M) 05 120583L of reverse primer (10 120583M) 107 120583L ofdistilledwater and 03120583L of cDNA template PCRwas carriedout by following the standard PCR program suggested by themanufacturerrsquos protocol usingMx3000P (Stratagene La JollaCA USA)

24 Data Analysis Scanned images were then imported intoGenePix Pro 60 software (Axon) for grid alignment and dataextraction Replicated miRNAs were averaged and miRNAswith intensitiesgt50 in all samples were chosen for calculatingnormalization factor Expressed data were normalized usingthemedian normalization After normalization differentiallyexpressed miRNAs were identified through volcano plotfiltering Hierarchical clustering was performed using MEVsoftware (v46 TIGR) GoStat was used to determine allgenes with statistically overrepresented gene ontology (GO)annotation [15]

3 Results

31 miRNA Expression Profiles in Gastric Cancer We usedmiRCURY array LNA miRNA chip which contains morethan 1891 capture probes to evaluate miRNA expressionprofiles between cancerous tissues and normal tissues Whensetting average change gt2-fold and 119875 value lt005 as a cut-offlevel 31 miRNAs are upregulated (Table 1) and 30 miRNAsare downregulated in gastric cancers (Table 2) Comparing

Gastroenterology Research and Practice 3

Table 1 List of upregulated expression miRNAs in gastric cancer

Gene name Fold change 119875 valuehsa-miR-105 34367019 00084407hsa-miR-4309 36494686 00061254hsa-miR-3664 32239638 00127328hsa-miR-187 73210044 00399018hsa-miR-4307 29131846 00054889hsa-miR-519e 32802326 00005427hsa-miR-631 47584436 00180121hsa-miR-491-5p 26810687 00411691hsa-miR-4278 77996863 00400128hsa-miR-548n 29842763 00338838hsa-miR-514b-3p 90201538 00210931hsa-miR-3920 42992771 00108692hsa-miR-376alowast 58401803 00390177hsa-miR-214lowast 77435982 00038199hsa-miRPlus-J1011 25587661 00436641hsa-miR-3158 52006402 00006224hsa-miR-1286 37748058 00156883hsa-let-7clowast 22461093 00126919hsa-miR-654-3p 31872897 00308998hsa-miR-1538 26964967 00355049hsa-miR-515-3p 35260133 00339529hsa-miR-2114 3390285 00027316hsa-miR-487a 21721574 00405062mcv-miR-M1-3p 2320875 00195074hsa-miR-2113 24646054 00439924hsa-miRPlus-C1114 27597195 00181086hsa-miR-133b 22714611 00404879hsa-miR-3670 34452207 00147389hsa-miR-491-3p 21276933 00162673hsa-miR-548aa 12978879 00303442hsa-miR-656 22199092 00019275

with previous studies of miRNAs we found that 21 among31 genes have been reported in previous publications forexample hsa-miR-105 hsa-miR-187 hsa-miR-214lowast hsa-miR-656 and hsa-miR-487a New upregulated genes includenamed genes such as hsa-miR-4309 hsa-miR-4307 hsa-miR-4278 and hsa-miRPlus-C1114 Twenty-six among 30downregulated genes have been reported before such as hsa-miR-31 hsa-miR-1275 hsa-miR-526b hsa-miR-2114 and hsa-miR-378c and hsa-miR-4303 hsv2-miR-H13 and hsv2-miR-H10were first reportedwith downregulation in gastric cancerWe provide new miRNA expression profiles of gastric cancer(Tables 1 and 2)

32 Quality Assessment of miRNA Data after Filtering Toassess miRNA data we built box plots to visualize thedistribution of the miRNA dataset Among six miRNA arraychips the distributions of log2 ratios are nearly the same(Figure 1(a)) Then we apply correlation matrix to elevatecorrelation among replicate experiments The scatter-plot

Table 2 List of downregulated expressionmiRNA in gastric cancer

Gene name Fold change 119875 valuehsa-miR-31 01444692 0004953hsa-miR-1275 03939808 0011642hsa-miR-26blowast 01534911 00189828hsa-miR-744 03825669 00009822hsa-miR-146b-5p 03483215 00237032hsa-miR-767-5p 04524493 00206438hsv2-miR-H13 03396123 0017379hsa-miR-526b 02088823 00086282ebv-miR-BART19-3p 04779629 00041381hsa-miR-518flowast 02507089 00171976hsa-miR-3196 03889389 00128937hsa-miR-3607-3p 04830454 00471262hsa-miR-542-3p 04221895 00009132hsa-miRPlus-A1087 04015859 00240223hsa-miR-518clowast 04395746 00160217hsv2-miR-H10 03909028 00087108hsa-miR-221 04285778 00077903kshv-miR-K12-4-3p 04321184 00215663hsa-miR-144 02929966 00320539hsa-miR-9 03090372 00120556hsa-miR-4303 04970407 3813119864 minus 05

hsa-miR-200c 03684039 00433953hsa-miR-3917 03883737 0000637hsa-miR-29c 0341262 00424996ebv-miR-BART6-3p 04265503 00434011hsa-miR-518elowast 04387169 0002632hsa-miR-141 0278258 0032224hsa-miR-34a 02989643 00172656hsa-miRPlus-D1058 02284739 0002042hsa-miR-378c 02607431 00395669

was used to assess the variation between cancer and normaltissues There are more than 80 of the same miRNA genesbetween cancerous and normal tissues (Figure 1(b))

33 Clustering Analysis of the Significantly Changed GenesTo identify differentially expressed miRNAs with statisticalsignificance we performed a volcano plot filtering betweenthe cancerous and normal miRNAs from the experiment(Figure 2(a)) The threshold we used to screen up- or down-regulated miRNAs is fold change ge20 and 119875 value lt005 Asshown in Figure 2(a) the red points in the plot represent thedifferentially expressed genes with statistical significance Inthis instance we identified 182 commonly expressed miRNAgenes From this set 31 were highly expressed and 30 showedlow levels of expression across the cancerous and normaltissues The following hierarchical clustering was performedbased on differentially expressed miRNA in cancerous versusnormal volcano plot The result of hierarchical clusteringshows distinguishable miRNA expression profiling amongcancerous and normal tissues (Figure 2(b)) We set the119875 value at lt005 as a cut-off level Expression levels of


14

12

10

8

6

4

2ra

tiolo

g

N1 N2 N3 C1 C2 C3

Before normalization

2ra

tiolo

g

N1 N2 N3 C1 C2 C3

After normalization6

4

2

0

minus2

minus4

minus6

(a)

Normal

Canc

er

1e minus 03

1e minus 02

1e minus 01

1e + 00

1e + 01

1e + 02

1e + 03

1e minus 03 1e minus 02 1e minus 01 1e + 00 1e + 01 1e + 02 1e + 03

(b)

Figure 1 (a)The box plots are used to compare distributions of samples (left nonnormalized log2-ratio data right median normalized log2-ratio data) (b)The scatter-plots assess the variation of miRNAs expression between cancerous and normal tissuesThe axes of the scatter-plotare the normalized signal values of the samples (ratio scale)

31 upregulated genes and 30 downregulated genes wereanalyzed by unsupervised hierarchical clustering Our datashow that all 61 miRNAs express similar patterns The heat-map demonstrated that all these genes changed similarly inthe different pairs of gastric cancerous and normal tissues(Figure 2(b)) The aberrantly expression miRNAs such ashas-miR-214lowast has-miR-105 has-miR-548n and has-miR-514which are upregulate in gastric cancerous tissues

34 Gene Ontology Analysis The differentially expressedmiRNAs would be expected to be significant to gastriccancer biology Gene ontology (GO) analysis was appliedto examine the significant ldquobiological processrdquo classificationsthat are overrepresented among these genes This analysis

revealed that many of the genes associated with tumor cellproliferationmainly take part in the processes of regulation ofcell proliferation andmaintain cell morphogenesis digestionand metabolism (Figure 3)

35 Validation of Aberrantly Expressed miRNA by Quantita-tive PCR Analysis To validate the findings from expressionarrays four miRNAs were tested by real-time quantitativePCR analysis We selected hsa-miR-105 hsa-miR-214lowast hsa-miR-514b and has-miR-548n to test in 24 paired gastricnormal and tumor tissues Control miRNA was U6 ForeverymiRNA that was upregulated expression bymicroarrayThe expression level of miR-214lowast is increased 379-fold(Figure 4(a)) miR-105 is increased 1632-fold (Figure 4(b))


Mean (cancer)-mean (normal)

00

05

10

15

20

25

30

35

40

45

minus5 minus4 minus3 minus2 minus1 0 1 2 3 4 5

minusLo

g10

(p)

(a)

hsa-miR-3670hsa-miR-2114hsa-miR-656hsa-miR-1538hsa-miR-487ahsa-miR-1286hsa-miR-4307mcv-miR-M1-3phsa-miR-3920hsa-miR-3158hsa-miR-105hsa-miR-4278hsa-miR-9hsa-miRPlus-D1058hsa-miR-526bhsa-miR-542-3phsa-miR-3917hsv2-miR-H13kshv-miR-K12-4-3phsa-miR-378chsa-miR-491-5phsa-miR-2113hsa-miR-133bhsa-miRPlus-J1011hsa-miR-3196hsv2-miR-H10hsa-miR-1275hsa-miR-31hsa-miR-144hsa-miR-4303hsa-miR-221hsa-miRPlus-A1087hsa-miR-34ahsa-miR-514b-3phsa-miR-187hsa-miR-548aahsa-miR-631hsa-miR-515-3phsa-miRPlus-C1114hsa-miR-3664hsa-miR-548nhsa-miR-4309hsa-miR-654-3phsa-miR-491-3phsa-miR-519eebv-miR-BART6-3phsa-miR-146b-5phsa-miR-3607-3phsa-miR-767-5phsa-miR-29chsa-miR-744ebv-miR-BART19-3p

hsa-miR-518flowast

hsa-let-7clowast

hsa-miR-518clowast

hsa-miR-214lowast

hsa-miR-26blowast

hsa-miR-518elowast

hsa-miR-200chsa-miR-141

hsa-miR-376alowast

CancerNormal

174

87

00

009491897

minus90 minus20 60

(b)

Figure 2 (a) Volcano plot showing the relative expression of miRNA genes form a one-class 119905-test The vertical lines correspond to 20-foldup and down respectively and the horizontal line represents a 119875 value of 005The red point in the plot represents the differentially expressedgenes with statistical significance (b) Hierarchical clustering for differentially expressed miRNAs in cancer versus normal pass volcano plot(fold change ge20) Red indicates high relative expression and green indicates low relative expression

has-miR-548 is 421-fold (Figure 4(c)) and miR-514 isincreased 1176-fold (Figure 4(d)) in comparing with normaltissuesThose are inconsistent with the result of miRNA chip

4 Discussion

Gastric cancer should be viewed as a heterogeneous diseaseshowing multiple biological and clinical differences Recentseveral studies have shown the dysregulation of somemiRNAin gastric cancer [16ndash18] Previous high-throughput arrayanalyses have clearly demonstrated that the miRNA expres-sion profile is substantially altered in cancer samples [19]

Herein we report comprehensive miRNA profiling in gastriccancer In this study we identified 61 miRNAs that areaberrantly expressed in gastric cancer Comparing with otherstudies we applied the microarray with the most hugedetectable miRNA library with 1891 capture probes As aresult a total of 31 miRNAs were upregulated in canceroustissues whereas 30 miRNAs were downregulated Usually weset miRNA expression change at 2-fold as a cut-off and thismay neglect aberrant miRNAs in the chip analysis Li et alhad established miRNA profiles of intestinal-type gastriccancer with 76 aberrantly expressedmiRNAs [6]ThemiRNAarray chip of this study detected those 76 miRNA capture


GO0042127regulationofcellproliferationGO0008283cellproliferationGO0008284positiveregulationofcellproliferationGO0048518positiveregulationofbiologicalprocessGO0009653anatomicalstructuremorphogenesisGO0007586digestionGO0044255cellularlipidmetabolicprocess

Figure 3 Clustering of overrepresented gene ontology (GO) classes in predicted targets of differential miRNAs

probes However only eight aberrantly expressed miRNAs(including has-miRplus-A1087 has-miR-542-3p has-miR-141 has-miR-200c has-miR-214 has-miR-29c has-miR-378and has-miR-128) were consistent with our findings Thereasons for discrepancy of two studies might be derived fromdifferent tumor types and heterogeneous On the other handmost ofmiRNAgenes in our studymight be newly detected ingastric cancer tissue and refer to important pathways duringprogress of carcinogenesis

Some of the differentially expressed miRNAs in gas-tric cancer were proved to be aberrantly by other studiesHelicobacter pylori (H pylori) infection is one of the mostprevalent infections worldwide and has been identified as themajor cause of this malignant disease A subset of miRNAreported to associate with H pylori infection such as has-miR-141 has-miR-146a and has-miR-2114 is detected in ourstudy The has-miR-2114 was reported to be upregulatedwhile has-miR-141 and has-miR-146a were downregulated[20] This finding coincided with our results (Tables 1 and2) Guo J et al identified 19 differential miRNAs in gastriccancer tissues Among them hsa-miR-31 and has-miR-133bwere includedwhichwere screened in our findings [21] In thepresent study the miRNA chip revealed that the has-miR-31was downregulated in gastric cancer tissues consistent witha previous study by Zhang et al which had identified thathas-miR-31 was lower in cancer tissues in comparison withnoncancerous tissues

The metastases and invasions are the most importantcharacteristics of malignant tumor A lot of miRNAs genesdetected in our study have been found to refer to thoseprogresses by other studies and might serve as biomarkersto reflect disease state has-miR-31 was frequently alteredin a large variety of cancers For example in breast cancerloss of has-miR-31 expression is associated with high riskmetastases [22] whereas in colorectal cancer high has-miR-31 expression correlates with advanced disease stage [23]In the present study has-miR-200c and has-miR-221 weredownregulated and consistent with previous research [24]which confirmed that the downregulation of has-miR-200cand has-miR-221 could serve as one diagnostic biomarker

of gastric cancer [25] The miR-187 was increased in breastcancer tissues which could be used as one of the independentprognostic factors and enforced tumor cells invasive ability[26] The accumulation of unedited has-miR-376alowast wasassociated with glioma tumor metastasis and promoted cellmigration and invasions [27] Wang et al proved that has-let-7clowast inhibited migration and invasion of non-small celllung cancer by targeting ITGB3 and MAP4K3 [28] Thep21 as tumor inhibitor had been explored the overexpres-sion of has-miR-515-3p rescued human mammary epithelialcells from Ras-induced senescence by prevention of Ras-induced upregulation of p21 [29] The alterations in miRNAexpression contributed to response chemotherapy The has-miR-487a could directly regulate breast cancer resistanceprotein (BCRP) expression and reverse chemotherapeuticdrug resistance in a subset of breast cancers [30] has-miR-546b-5p had inhibitory effect on pancreatic cancer cellmigration and invasion by targeting MMP16 [31]

Expression levels of four miRNAs were analyzed byquantitative real-time PCR The results are consistent withmiRNAs chip All of them were upregulated in gastriccancerous tissues The has-miR-105 was highly expressedin testis tumors [32] The inhibitor efforts of has-miR-214had been reported in several types of tumors includinghepatocellular carcinoma cholangiocarcinoma and ovariancancer [33ndash35] has-miR-214 had multiple roles in regulatingtumor cell characteristics such as proliferation andmigrationby targeting p53 and 120573-catenin [36] Only one report byWotschofsky et al detected has-miR-514b and identifiedthat the expression of has-miR-514b was downregulated inprimary metastatic renal cell carcinoma [37] At last theexpression level of has-miR-548n was upregulated in gastriccancer The aberrantly of has-miR-548n may be associatedwith host antiviral response via direct targeting of interferon-120574 [38]

By targeting tens to hands of genes miRNAs can redirectbasic biological functions and pathway essential to tumordevelopment and progression According to GO analysis themajor roles of aberrantly expressed miRNA regulated cellproliferation (Figure 3)The aberrantly expressed miRNAs of


Normal Cancer00

25

50

75

100

125

150

Relat

ive e

xpre

ssio

n of

miR

NA

has-miR-214lowast

(a)

Normal Cancer

Relat

ive e

xpre

ssio

n of

miR

NA

0

5

10

15

20

25

30

35

40

45

50

hsa-miR-105

(b)

Normal Cancer

Relat

ive e

xpre

ssio

n of

miR

NA

0

2

4

6

8

10has-miR-548

(c)

Normal Cancer

Relat

ive e

xpre

ssio

n of

miR

NA

0

25

50

75

100

125

150

175

200

has-miR-514

(d)

Figure 4ThemiRNA expression level of has-miR-214lowast (a) has-miR-105 (b) has-miR-548n (c) and has-miR-514b (d) detected by qRT-PCRin cancerous and normal tissues Relative gene expression was calculated as 2minusΔΔCT

gastric cancer had been associated with regulating cell Wedetected the downregulation of has-miR-9 in gastric cancerwhich suppressed the proliferation invasion and metastasisof gastric cancer cells through targeting cyclin D1 and Ets1[39] AnothermiRNAgenemiR-144 was downregulated andhad reported increased bladder cancer cell proliferation bytargeting EZH2 and regulating Wnt signaling [40] Further-moremiRNAs served asmediators of inflammation in tumorprogression through regulation of components of immunesystem For example the high levels of has-miR-187 promotedlymph node metastasis of breast cancer [26]

We also found some new aberrant expression miRNAsin gastric cancers (Tables 1 and 2) which have no or very

few reports of aberrant expression in any other cancersThese new aberrant miRNAs are dysregulated in gastriccancer ant the impact on gene expression As more miRNAsare identified and validated the role of aberrant miRNAexpression in gastric cancer will be better understood Ourdata may provide diagnostic or prognostic biomarkers ofgastric cancer and offer new molecular targets for therapy ofgastric cancer

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper


Acknowledgments

This work was supported by Scientific Research Foundationof The Affiliated Tumor Hospital of Harbin Medical Uni-versity (Grant no JJZ2010-05) Scientific Research Subjectof Health Department of Heilongjiang Province of China(Grant no 2011-130) Natural Science Foundation of Hei-longjiang Province of China General Program (Grant noD201120) All authors have contributed significantly and allauthors are in agreement with the content of the paper

References

[1] R Siegel D Naishadham and A Jemal ldquoCancer statisticsrdquo CAA Cancer Journal for Clinicians vol 63 pp 11ndash30 2013

[2] Y Lin J Ueda S Kikuchi et al ldquoComparative epidemiologyof gastric cancer between Japan and Chinardquo World Journal ofGastroenterology vol 17 no 39 pp 4421ndash4428 2011

[3] H Tang Y Kong J Guo et al ldquoDiallyl disulfide suppressesproliferation and induces apoptosis in human gastric cancerthroughWnt-1 signaling pathway by up-regulation ofmiR-200band miR-22rdquo Cancer Letters vol 340 no 1 pp 72ndash81 2013

[4] C Li J F Li Q Cai et al ldquoMiRNA-199a-3p a potential circu-lating diagnostic biomarker for early gastric cancerrdquo Journal ofSurgical Oncology vol 108 pp 89ndash92 2013

[5] B Mınguez and A Lachenmayer ldquoDiagnostic and prognosticmolecular markers in hepatocellular carcinomardquoDiseaseMark-ers vol 31 no 3 pp 181ndash190 2011

[6] X Li F Luo Q Li et al ldquoIdentification of new aberrantlyexpressed miRNAs in intestinal-type gastric cancer and itsclinical significancerdquo Oncology Reports vol 26 no 6 pp 1431ndash1439 2011

[7] S P Nana-Sinkam and C M Croce ldquoClinical applications formicroRNAs in cancerrdquoClinical Pharmacology andTherapeuticsvol 93 pp 98ndash104 2013

[8] Y Shimono M Zabala R W Cho et al ldquoDownregulation ofmiRNA-200c links breast cancer stem cells with normal stemcellsrdquo Cell vol 138 no 3 pp 592ndash603 2009

[9] P Trang J F Wiggins C L Daige et al ldquoSystemic deliveryof tumor suppressor microRNA mimics using a neutral lipidemulsion inhibits lung tumors in micerdquoMolecularTherapy vol19 no 6 pp 1116ndash1122 2011

[10] S Volinia M Galasso S Costinean et al ldquoReprogramming ofmiRNA networks in cancer and leukemiardquo Genome Researchvol 20 no 5 pp 589ndash599 2010

[11] W Cao R Fan L Wang et al ldquoExpression and regulatoryfunction of miRNA-34a in targeting survivin in gastric cancercellsrdquo Tumour Biology vol 34 pp 963ndash971 2013

[12] Y Yao A Suo Z Li et al ldquoMicroRNAprofiling of human gastriccancerrdquo Molecular Medicine Reports vol 2 no 6 pp 963ndash9702009

[13] D Liu P Xia D Diao et al ldquoMiRNA-429 suppresses the growthof gastric cancer cells in vitrordquo Journal of Biomedical Researchvol 26 pp 389ndash393 2012

[14] F Wang T Li B Zhang et al ldquoMicroRNA-19ab regulatesmultidrug resistance in human gastric cancer cells by targetingPTENrdquo Biochemical and Biophysical Research Communicationsvol 434 pp 688ndash694 2013

[15] T Beissbarth ldquoInterpreting experimental results using geneontologiesrdquoMethods in Enzymology vol 411 pp 340ndash352 2006

[16] T S Yang X H Yang X D Wang Y L Wang B Zhou andZ S Song ldquoMiR-214 regulate gastric cancer cell proliferationmigration and invasion by targeting PTENrdquo Cancer Cell Inter-national vol 13 p 68 2013

[17] B Jiang Z Li W Zhang et al ldquomiR-874 Inhibits cell prolifera-tion migration and invasion through targeting aquaporin-3 ingastric cancerrdquo Journal of Gastroenterology In press

[18] X Liu J Ru J Zhang et al ldquomiR-23a targets interferonregulatory factor 1 and modulates cellular proliferation andpaclitaxel-induced apoptosis in gastric adenocarcinoma cellsrdquoPLoS ONE vol 8 Article ID e64707 2013

[19] CWong C CWong JM Lee DN Fan S L Au and I ONgldquoSequential alterations ofmicrorna expression in hepatocellularcarcinoma development and venous metastasisrdquo Hepatologyvol 55 no 5 pp 1453ndash1461 2012

[20] T Nishizawa and H Suzuki ldquoThe role of microRNA in gastricmalignancyrdquo International Journal of Molecular Sciences vol 14pp 9487ndash9496 2013

[21] J Guo Y Miao B Xiao et al ldquoDifferential expression ofmicroRNA species in human gastric cancer versus non-tumorous tissuesrdquo Journal of Gastroenterology and Hepatologyvol 24 no 4 pp 652ndash657 2009

[22] Y Zhang J Guo D Li et al ldquoDown-regulation of miR-31expression in gastric cancer tissues and its clinical significancerdquoMedical Oncology vol 27 no 3 pp 685ndash689 2010

[23] E M Laurila and A Kallioniemi ldquoThe diverse role of miR-31 inregulating cancer associated phenotypesrdquo Genes Chromosomesand Cancer vol 52 no 12 pp 1103ndash1113 2013

[24] B Q Yu L P Su J F Li et al ldquomicroRNA expression signatureof gastric cancer cells relative to normal gastric mucosardquoMolecular Medicine Reports vol 6 pp 821ndash826 2012

[25] H Tang M Deng Y Tang et al ldquomiR-200b and miR-200cas prognostic factors and mediators of gastric cancer cellprogressionrdquo Clinical Cancer Research vol 19 no 20 pp 5602ndash5612 2013

[26] L Mulrane S F Madden D J Brennan et al ldquomiR-187 isan independent prognostic factor in breast cancer and confersincreased invasive potential in vitrordquo Clinical Cancer Researchvol 18 pp 6702ndash6713 2012

[27] YChoudhury F C TayDH Lamet al ldquoAttenuated adenosine-to-inosine editing of microRNA-376alowast promotes invasivenessof glioblastoma cellsrdquo Journal of Clinical Investigation vol 122pp 4059ndash4076 2012

[28] B Zhao H Han J Chen et al ldquomicroRNA let-7c inhibitsmigration and invasion of human non-small cell lung cancerby targeting ITGB3 and MAP4K3rdquo Cancer Letters vol 342 no1 pp 43ndash51 2013

[29] V Borgdorff M E Lleonart C L Bishop et al ldquoMultiplemicroRNAs rescue from Ras-induced senescence by inhibitingp21 (Waf1Cip1)rdquoOncogene vol 29 no 15 pp 2262ndash2271 2010

[30] M T Ma M He Y Wang et al ldquoMiR-487a resensitizes mitox-antrone (MX)-resistant breast cancer cells (MCF-7MX) to MXby targeting breast cancer resistance protein (BCRPABCG2)rdquoCancer Letters vol 339 pp 107ndash115 2013

[31] F Lin X Wang Z Jie et al ldquoInhibitory effects of miR-146b-5p on cell migration and invasion of pancreatic cancer bytargeting MMP16rdquo Journal of Huazhong University of Scienceand TechnologymdashMedical Science vol 31 no 4 pp 509ndash5142011

[32] G W Novotny K C Belling J B Bramsen et al ldquoMicroRNAexpression profiling of carcinoma in situ cells of the testisrdquoEndocrine-Related Cancer vol 19 pp 365ndash379 2012


[33] T C Shih Y J Tien C JWen et al ldquoMicroRNA-214 downregu-lation contributes to tumor angiogenesis by inducing secretionof the hepatoma-derived growth factor in human hepatomardquoJournal of Hepatology vol 57 pp 584ndash591 2012

[34] B Li Q Han Y Zhu Y Yu J Wang and X Jiang ldquoDown-regulation of miR-214 contributes to intrahepatic cholangiocar-cinoma metastasis by targeting Twistrdquo FEBS Journal vol 279pp 2393ndash2398 2012

[35] C X Xu M Xu L Tan et al ldquoMicroRNA miR-214 regulatesovarian cancer cell stemness by targeting p53Nanogrdquo TheJournal of Biological Chemistry vol 287 pp 34970ndash34978 2012

[36] X Wang J Chen F Li et al ldquoMiR-214 inhibits cell growth inhepatocellular carcinoma through suppression of beta-cateninrdquoBiochemical andBiophysical ResearchCommunications vol 428pp 525ndash531 2012

[37] Z Wotschofsky J Busch M Jung et al ldquoDiagnostic and prog-nostic potential of differentially expressed miRNAs betweenmetastatic and non-metastatic renal cell carcinoma at the timeof nephrectomyrdquo Clinica Chimica Acta vol 416 pp 5ndash10 2013

[38] Y Li J Xie X Xu et al ldquoMicroRNA-548 down-regulates hostantiviral response via direct targeting of IFN-lambda1rdquo Proteinamp Cell vol 4 pp 130ndash141 2013

[39] L Zheng T Qi D Yang et al ldquomicroRNA-9 suppresses theproliferation invasion and metastasis of gastric cancer cellsthrough targeting cyclin D1 and Ets1rdquo PLoS ONE vol 8 ArticleID e55719 2013

[40] Y Guo L Ying Y Tian et al ldquomiR-144 downregulationincreases bladder cancer cell proliferation by targeting EZH2and regulatingWnt signalingrdquo FEBS Journal vol 280 pp 4531ndash4538 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom 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 httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


miRNAs in gastric cancer miR-199a-3p is significantly higherin serum of gastric cancer patients and may be used asdiagnostic biomarker [4] MiR-34a was downregulated ingastric cancer cell lines [11 12] MiR-429 suppresses tumorcells proliferation and may serve as tumor suppressor duringtumorigenesis of gastric cancer [13] MiR-19ab regulatesmultidrug resistance in gastric cancer by targeting PTEN [14]Li et al had identified abnormal expressed miRNA profileswith 40 upregulatedmiRNAs and 36 downregulatedmiRNAsin intestinal-type gastric caners by miRNA array [6] It hasbeen estimated that there are about 1000 miRNAs in humanbut previous studies have only screened miRNA expressionin gastric cancer patients from less than 500 miRNAs It isnecessary to screen gastric cancer with larger collections ofmiRNAs by gene chip

To explore more related miRNAs we have used 6thgeneration of miRNA array that contains more than 1891probes which include nearly all human miRNAs and identi-fied new aberrant expressionmiRNAs between gastric cancerand normal tissues We identified 61 new obviously changedexpression miRNAs in gastric cancers Our data offer newclues to study the miRNA profiles that refer to molecularmechanism of gastric cancer carcinogenesis and providenovel biomarkers repertoire of this malignant disease

2 Material and Methods

The tissue samples in this study were derived from patientsundergoing a surgical procedure to remove a portion ofgastric cancer at the Affiliated Tumor Hospital of HarbinMedical University The collection of samples conformed tothe policies of China and practices of the facilityrsquos Institu-tional Review Board Upon removal of the surgical specimenresearch personnel immediately transported the tissue to thesurgical pathology laboratory Pathology faculty performeda gross analysis of the specimen and selected cancerousappearing gastric tissue and normal appearing gastric cancerfor research Each sample was placed in a cryovial andpreserved in minus80∘C refrigerator until analysis Subsequentlythe surgical specimens confirmed the histopathology of thesamples taken for research

21 Total RNA Isolation and Quality Analysis Frozen tissueswere first pulverized in a stainless steel mortar and pestleTotal RNA was isolated using TRIzol (Invitrogen CarslbadCA) and miRNeasy mini kit (QIAGEN) according to man-ufacturerrsquos instructions RNA quality and quantity weremeasured by using nanodrop spectrophotometer (ND-1000Nanodrop Technologies) and RNA integrity was determinedby gel electrophoresis

22 miRNA Precursor Expression Profiling After RNA iso-lation from the samples the miRCURY Hy3Hy5 powerlabeling kit (Exiqon Vedbaek Denmark) was used accordingto the manufacturerrsquos guideline for miRNA labelling Onemicrogram of each sample was 31015840-end-labeled with Hy3fluorescent label using T4 RNA ligase by the followingprocedure RNA in 20 120583L of water was combined with 10 120583L

of CIP buffer and CIP (Exiqon) The mixture was incubatedfor 30min at 37∘C and was terminated by incubation for5min at 95∘C Then 30 120583L of labeling buffer 15 120583L offluorescent label (Hy3) 20120583L of DMSO and 20 120583L oflabeling enzyme were added into the mixture The labelingreaction was incubated for 1 h at 16∘C and terminated byincubation for 15min at 65∘C After stopping the labelingprocedure the Hy3-labeled samples were hybridized onthe miRCURY LNA array (v160) (Exiqon) according toarray manual The total 25 120583L mixture from Hy3-labeledsamples with 25 120583L hybridization buffer was first denaturedfor 2min at 95∘C incubated on ice for 2min and thenhybridized to the microarray for 16ndash20 h at 56∘C in a 12-Bay Hybridization Systems (Hybridization System Nimble-gen Systems Inc Madison WI USA) which provides anactive mixing action and constant incubation temperatureto improve hybridization uniformity and enhance signalFollowing hybridization the slides were achieved washedseveral times usingWash buffer kit (Exiqon) and finally driedby centrifugation for 5min at 400 rpm Then the slides werescanned using the Axon GenePix 4000B microarray scanner(Axon Instruments Foster City CA)

23 miRNA Quantification by Real-Time RT-PCR (qRT-PCR)SYBRGreen RT-qPCR assay was used for miRNA quantifica-tion In brief one microgram of extracted RNA was reverse-transcribed per the manufacturerrsquos instructions qRT-PCRwas carried out in thin-wall PCR plates (Applied BiosystemsFoster City CA USA) Each reaction mixture contained125 120583L of SYBR Premix Ex Taq (2times) 05 lL of referencedye (ROX) II (50times) (Takara Otsu Japan) 05 120583L of forwardprimer (10120583M) 05 120583L of reverse primer (10 120583M) 107 120583L ofdistilledwater and 03120583L of cDNA template PCRwas carriedout by following the standard PCR program suggested by themanufacturerrsquos protocol usingMx3000P (Stratagene La JollaCA USA)

24 Data Analysis Scanned images were then imported intoGenePix Pro 60 software (Axon) for grid alignment and dataextraction Replicated miRNAs were averaged and miRNAswith intensitiesgt50 in all samples were chosen for calculatingnormalization factor Expressed data were normalized usingthemedian normalization After normalization differentiallyexpressed miRNAs were identified through volcano plotfiltering Hierarchical clustering was performed using MEVsoftware (v46 TIGR) GoStat was used to determine allgenes with statistically overrepresented gene ontology (GO)annotation [15]

3 Results

31 miRNA Expression Profiles in Gastric Cancer We usedmiRCURY array LNA miRNA chip which contains morethan 1891 capture probes to evaluate miRNA expressionprofiles between cancerous tissues and normal tissues Whensetting average change gt2-fold and 119875 value lt005 as a cut-offlevel 31 miRNAs are upregulated (Table 1) and 30 miRNAsare downregulated in gastric cancers (Table 2) Comparing












14

12

10

8

6

4

2ra

tiolo

g

N1 N2 N3 C1 C2 C3


2ra

tiolo

g

N1 N2 N3 C1 C2 C3


4

2

0

minus2

minus4

minus6

(a)

Normal

Canc

er

1e minus 03

1e minus 02

1e minus 01

1e + 00

1e + 01

1e + 02

1e + 03


(b)








00

05

10

15

20

25

30

35

40

45


minusLo

g10

(p)

(a)


hsa-miR-518flowast

hsa-let-7clowast

hsa-miR-518clowast

hsa-miR-214lowast

hsa-miR-26blowast

hsa-miR-518elowast


hsa-miR-376alowast

CancerNormal

174

87

00

009491897

minus90 minus20 60

(b)



4 Discussion













Normal Cancer00

25

50

75

100

125

150

Relat

ive e

xpre

ssio

n of

miR

NA

has-miR-214lowast

(a)

Normal Cancer

Relat

ive e

xpre

ssio

n of

miR

NA

0

5

10

15

20

25

30

35

40

45

50

hsa-miR-105

(b)

Normal Cancer

Relat

ive e

xpre

ssio

n of

miR

NA

0

2

4

6

8

10has-miR-548

(c)

Normal Cancer

Relat

ive e

xpre

ssio

n of

miR

NA

0

25

50

75

100

125

150

175

200

has-miR-514

(d)








Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























14

12

10

8

6

4

2ra

tiolo

g

N1 N2 N3 C1 C2 C3


2ra

tiolo

g

N1 N2 N3 C1 C2 C3


4

2

0

minus2

minus4

minus6

(a)

Normal

Canc

er

1e minus 03

1e minus 02

1e minus 01

1e + 00

1e + 01

1e + 02

1e + 03


(b)








00

05

10

15

20

25

30

35

40

45


minusLo

g10

(p)

(a)


hsa-miR-518flowast

hsa-let-7clowast

hsa-miR-518clowast

hsa-miR-214lowast

hsa-miR-26blowast

hsa-miR-518elowast


hsa-miR-376alowast

CancerNormal

174

87

00

009491897

minus90 minus20 60

(b)



4 Discussion













Normal Cancer00

25

50

75

100

125

150

Relat

ive e

xpre

ssio

n of

miR

NA

has-miR-214lowast

(a)

Normal Cancer

Relat

ive e

xpre

ssio

n of

miR

NA

0

5

10

15

20

25

30

35

40

45

50

hsa-miR-105

(b)

Normal Cancer

Relat

ive e

xpre

ssio

n of

miR

NA

0

2

4

6

8

10has-miR-548

(c)

Normal Cancer

Relat

ive e

xpre

ssio

n of

miR

NA

0

25

50

75

100

125

150

175

200

has-miR-514

(d)








Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















14

12

10

8

6

4

2ra

tiolo

g

N1 N2 N3 C1 C2 C3


2ra

tiolo

g

N1 N2 N3 C1 C2 C3


4

2

0

minus2

minus4

minus6

(a)

Normal

Canc

er

1e minus 03

1e minus 02

1e minus 01

1e + 00

1e + 01

1e + 02

1e + 03


(b)








00

05

10

15

20

25

30

35

40

45


minusLo

g10

(p)

(a)


hsa-miR-518flowast

hsa-let-7clowast

hsa-miR-518clowast

hsa-miR-214lowast

hsa-miR-26blowast

hsa-miR-518elowast


hsa-miR-376alowast

CancerNormal

174

87

00

009491897

minus90 minus20 60

(b)



4 Discussion













Normal Cancer00

25

50

75

100

125

150

Relat

ive e

xpre

ssio

n of

miR

NA

has-miR-214lowast

(a)

Normal Cancer

Relat

ive e

xpre

ssio

n of

miR

NA

0

5

10

15

20

25

30

35

40

45

50

hsa-miR-105

(b)

Normal Cancer

Relat

ive e

xpre

ssio

n of

miR

NA

0

2

4

6

8

10has-miR-548

(c)

Normal Cancer

Relat

ive e

xpre

ssio

n of

miR

NA

0

25

50

75

100

125

150

175

200

has-miR-514

(d)








Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















00

05

10

15

20

25

30

35

40

45


minusLo

g10

(p)

(a)


hsa-miR-518flowast

hsa-let-7clowast

hsa-miR-518clowast

hsa-miR-214lowast

hsa-miR-26blowast

hsa-miR-518elowast


hsa-miR-376alowast

CancerNormal

174

87

00

009491897

minus90 minus20 60

(b)



4 Discussion













Normal Cancer00

25

50

75

100

125

150

Relat

ive e

xpre

ssio

n of

miR

NA

has-miR-214lowast

(a)

Normal Cancer

Relat

ive e

xpre

ssio

n of

miR

NA

0

5

10

15

20

25

30

35

40

45

50

hsa-miR-105

(b)

Normal Cancer

Relat

ive e

xpre

ssio

n of

miR

NA

0

2

4

6

8

10has-miR-548

(c)

Normal Cancer

Relat

ive e

xpre

ssio

n of

miR

NA

0

25

50

75

100

125

150

175

200

has-miR-514

(d)








Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























Normal Cancer00

25

50

75

100

125

150

Relat

ive e

xpre

ssio

n of

miR

NA

has-miR-214lowast

(a)

Normal Cancer

Relat

ive e

xpre

ssio

n of

miR

NA

0

5

10

15

20

25

30

35

40

45

50

hsa-miR-105

(b)

Normal Cancer

Relat

ive e

xpre

ssio

n of

miR

NA

0

2

4

6

8

10has-miR-548

(c)

Normal Cancer

Relat

ive e

xpre

ssio

n of

miR

NA

0

25

50

75

100

125

150

175

200

has-miR-514

(d)








Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Normal Cancer00

25

50

75

100

125

150

Relat

ive e

xpre

ssio

n of

miR

NA

has-miR-214lowast

(a)

Normal Cancer

Relat

ive e

xpre

ssio

n of

miR

NA

0

5

10

15

20

25

30

35

40

45

50

hsa-miR-105

(b)

Normal Cancer

Relat

ive e

xpre

ssio

n of

miR

NA

0

2

4

6

8

10has-miR-548

(c)

Normal Cancer

Relat

ive e

xpre

ssio

n of

miR

NA

0

25

50

75

100

125

150

175

200

has-miR-514

(d)








Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article Identification of Aberrantly Expressed miRNAs in …downloads.hindawi.com/journals/grp/2014/473817.pdf · 2019. 7. 31. · Research Article Identification of Aberrantly