文献3 (2).pdf

HEPATOBILIARY MALIGNANCIESLong Noncoding RNA Associated With MicrovascularInvasion in Hepatocellular Carcinoma PromotesAngiogenesis and Serves as a Predictor forHepatocellular Carcinoma Patients PoorRecurrence-Free Survival After HepatectomySheng-Xian Yuan,1*Fu Yang,2*Yuan Yang,1Qi-Fei Tao,1Jin Zhang,1Gang Huang,1Yun Yang,1Ruo-Yu Wang,1Sen Yang,1Xi-Song Huo,2Ling Zhang,2Fang Wang,2Shu-Han Sun,2and Wei-Ping Zhou1Survival of patients with hepatocellular carcinoma(HCC)remains poor,which is largelyattributed to active angiogenesis.However,the mechanisms underlying angiogenesis inHCC remain to be discovered.In this study,we found that long noncoding RNA associ-ated with microvascular invasion in HCC(lncRNA MVIH)(lncRNA associated with mi-crovascular invasion in HCC)was generally overexpressed in HCC.In a cohort of 215HCC patients,the overexpression of MVIH was associated with frequent microvascularinvasion(P 5 0.016)and a higher tumor node metastasis stage(P 5 0.009)as well asdecreased recurrence-free survival(RFS)(P 0.001)and overall survival(P 5 0.007).Moreover,the up-regulation of MVIH served as an independent risk factor to predict poorRFS.We also found that MVIH could promote tumor growth and intrahepatic metastasisby activating angiogenesis in mouse models.Subsequent investigations indicated thatMVIH could activate tumor-inducing angiogenesis by inhibiting the secretion of phospho-glycerate kinase 1(PGK1).Additionally,in 65 HCC samples,MVIH expression was inver-sely correlated with the serum level of PGK1 and positively correlated with the microvesseldensity.Conclusion:Deregulation of lncRNA MVIH is a predictor for poor RFS of HCCpatients after hepatectomy and could be utilized as a potential target for new adjuvanttherapies against active angiogenesis.(HEPATOLOGY2012;56:2231-2241)Hepatocellular carcinoma(HCC)is currentlythe fifth-most common solid tumor world-wide and the second leading cause of cancer-related deaths in China.1,2Although remarkable pro-gress has been made in recent decades,the details ofthe molecular mechanisms underlying HCC carcino-genesis remain to be elucidated.2,3Survival of patientswith HCC has been improved with advancements insurgical techniques,but the median survival rateremains at approximately 50%(range,17-69)after 5years.4This unfavorable prognosis is mainly becauseHCC is a highly vascularized type of tumor with fre-quent intra-or extrahepatic metastases.Blood vesselswithin tumors produced by angiogenesis are responsi-ble for the poor survival of HCC patients.3,5Cancerclassification using biomarkers may effectively definerisk of recurrence,which allows for the use of appro-priate treatments to acquire a better prognosis.6But,to date,few measurable biomarkers for predictingHCC recurrence have been identified.Abbreviations:Ab,antibody;AFP,alpha-fetoprotein;BCLC,Barcelona Cancer Liver Clinic;CI,confidence interval;CSF,codon substitution frequency;ELISA,enzyme-linked immunosorbent assay;HBV,hepatitis B virus;HCC,hepatocelluar carcinoma;H&E,hematoxylin and eosin;HRs,hazard ratios;HUVECs,humanumbilical vein endothelial cells;IHC,immunohistochemistry;lncRNAs,long noncoding RNAs;miRNAs,microRNAs;MS,mass spectrometry;MVD,microvesseldensity;MVIH,lncRNAs associated with microvascular invasion in HCC;ORFs,open reading frames;OS,overall survival;PGK1,phosphoglycerate kinase 1;qRT-PCR,quantitative real-time polymerase chain reaction;RFS,recurrence-free survival;RIP,radioimmunoprecipitation;SC,subcutaneous;SDS-PAGE,sodiumdodecyl sulfate/polyacrylamide gel electrophoresis;siRNAs,short interfering RNAs;TNM,tumor node metastasis.From the1The Third Department of Hepatic Surgery,Eastern Hepatobiliary Surgery Hospital,Second Military Medical University,Shanghai,China;and2The Department of Medical Genetics,Second Military Medical University,Shanghai,China.Received January 31,2012;accepted May 30,2012.This work was supported by the Chinese Key Project for Infectious Diseases(2008ZX10002-018 and 2008ZX10002-025),the Science Fund for CreativeResearch Groups,National Natural Science Foundation of China(NSFC;grant no.:30921006),and the NSFC(grant nos.:81071680 and 30671920).*These authors contributed equally to this work.2231Long noncoding RNAs(lncRNAs)represent a sub-group of noncoding RNAs that are longer than 200nucleotides.Recently,studies have shown that moregenomic sequence is transcribed into lncRNAs thanprotein-coding RNAs.7,8Additionally,multiple studieshave indicated that significant numbers of lncRNAsare involved and might play central roles in a varietyof biological processes through complicated mecha-nisms.9-13Notably,the deregulation of lncRNAs hasalso been shown to result in aberrant gene expressionthat contributes to the progression of a variety ofhuman tumors,including HCC.14-17However,com-pared with protein-coding genes,the clinical signifi-cance and functions of most deregulated lncRNAs inthe progression and aggressiveness of HCC remainunknown.In the present study,we report an lncRNA termedMVIH(lnc RNAs associated with microvascular inva-sion in HCC)(NCBI no.:AK094613)that is up-regu-lated in tumor tissues,compared to the correspondingnoncancerous tissues,and correlates with the microvas-cular invasion of HCC.Moreover,MVIH serves as anindependent risk factor for HCC patients poor recur-rence-freesurvival(RFS)afterhepatectomy.Ourresults indicate that MVIH could promote tumorgrowth and intrahepatic metastasis in vivo.Further-more,we demonstrate that the inhibition of phospho-glycerate kinase 1(PGK1)secretion by its associationwith MVIH contributes to active angiogenesis bothin vitro and in vivo.Patients and MethodsPatients and Clinical Samples.Forty fresh HCCtissues and paired adjuvant noncancerous tissue sam-ples were randomly selected from patients undergoinghepatectomy at the Eastern Hepatobiliary SurgeryHospital(Shanghai,China)between May 1 and June30,2009.These tissues were used for quantitative real-time polymerase chain reaction(qRT-PCR)analysis.Another 215 fresh HCC tissues were collected fromthe same bank1and used for survival analysis and fur-ther validation.Fresh tissue and serum samples werecollected in the operating room and processed imme-diately within 15 minutes.Each sample was frozenand stored at?80?C.Paired noncancerous tissueswere isolated from at least 2 cm away from the tumorborder and were shown to lack tumor cells by micros-copy.All patients in this study met the followinginclusion criteria:Resected nodules were identified asHCC by pathological examination;no anticancer treat-ments were given before surgery;complete resection ofall tumor nodules was verified by the cut surface beingfreeofcancerbypathologicalexamination;andcomplete clinical-pathologic and follow-up data wereavailable.Patients that died of nonliver diseases oraccidents were excluded.Clinical characteristics of allpatients are listed in Supporting Table 1.Tumor differ-entiation was defined according to Edmondsons grad-ing system.2Micrometastases were defined as tumorsadjacent to the border of the main tumor that wereonly observed under the microscope.Tumor stagingwas defined according to the sixth edition of the tu-mor node metastasis(TNM)classification system pub-lished by the International Union Against Cancer andthe Barcelona Clinic Liver Cancer(BCLC)staging sys-tem.The study was approved by the institutionalreview board of Eastern Hepatobiliary Surgery Hospi-tal.All patients gave their written informed consent toparticipate in the study.The data do not contain anyinformation that could identify patients.Detailed description of Patients and Methods can befound in the online Supporting Materials.ResultsThe Novel lncRNA MVIH Is Up-regulated inHCC.We previously identified systemic variations inthe expression of lncRNAs between hepatitis B virus(HBV)-related HCC and paired nontumor samplesusing a microarray analysis.14From that study,we notedthat lncRNA MVIH was remarkably up-regulated(foldchange,3.654;P 0.00205)in HCC according to themicroarray data.(The microarray data discussed in thatarticle have been deposited in NCBI Gene ExpressionOmnibus and are accessible through GEO Series acces-sion number GSE27462;,http:/www.ncbi.nlm.nih.gov/geo/query/acc.cgi?accGSE 27462.)To further validatethis result,we analyzed MVIH expression using qRT-Address reprint requests to:Wei-Ping Zhou,M.D.,Ph.D,The Third Department of Hepatic Surgery,Eastern Hepatobiliary Surgery Hospital,225 ChanghaiRoad,200438 Shanghai,China.E-mail:;fax:86 021-81875529 or Shu-Han Sun,Ph.D.Department of Medical Genetics,SecondMilitary Medical University,800 Xiangyin Road,Shanghai 200433,P.R.China.E-mail:.CopyrightVC2012 by the American Association for the Study of Liver Diseases.View this article online at .DOI 10.1002/hep.25895Potential conflict of interest:Nothing to report.Additional Supporting Information may be found in the online version of this article.2232YUAN ET AL.HEPATOLOGY,December 2012PCR in 40 pairs of predominately HBV-related HCCand the corresponding peritumoral tissues(SupportingTable 1,cohort 1)and found that MVIH was significantlyup-regulated(P 0.007)in HCC(Fig.1A).We alsonoted that MVIH was located within the intron of the ri-bosomal protein S24(RPS24)gene and overlapped withpart of the 30end of the RPS24 transcript(Fig.1B).Toexplore the potential relationship of the MVIH andRPS24 transcripts,we first examined the expression levelsof RPS24 and MVIH in 40 HBV/HCC tissues(cohort1).The results showed that no correlation(r 0.060;P 0.695)existed between the transcriptional levels ofRPS24 and MVIH(Fig.1C).Furthermore,lncRNAMVIH was statistically unchanged in SMMC-7721 andHCCLM3 cells transfected with two different short inter-fering(siRNAs)(designated as si-1 and si-2)againstRPS24,despite significant reduction in RPS24 messengerRNA expression(Fig.1D).Next,we performed a rapidamplification of complementary DNA ends(RACE)anal-ysis to identify the 50and 30ends of the MVIH transcript.To amplify MVIH in a specific manner,we used intronicprimers that would not recognize RPS24(Fig.1B).Thefull sequence of lncRNA MVIH is presented in Support-ing Fig.1.To determine whether the transcript of MVIHmight encode proteins,we first predicted open readingframes(ORFs)of MVIH,and using an ORF finder fromthe National Center for Biotechnology Information(Be-thesda,MD),several ORFs were predicted from lncRNAMVIH,and only one showed homologous protein with56%identity in human.Second,we used a codon substi-tution frequency(CSF)analysis using PhyloCSF.Wefound that lncRNA MVIH had a very low CSF score(Supporting Fig.2),which indicated that the transcript oflncRNA MVIH had no protein-coding potential.In con-clusion,the overexpressed lncRN MVIH in HCC is anoncoding RNA and is transcribed independently of theRPS24 gene.The Association of lncRNA MVIH With Clinico-pathologicalCharacteristicsandPrognosisAfterHepatectomy.We next sought to determine whetherFig.1.lncRNA MVIH is up-regulated in HCC and transcribed independently of RPS24.(A)lncRNA MVIH is significantly up-regulated in 40human HCC tissues,compared to the corresponding noncancerous tissues.Statistical differences were analyzed using the paired t test.Horizon-tal lines in box plots represent the median,boxes represent the interquartile range,and whiskers represent the 2.5th and 97.5th percentiles.(B)Schematic representation of the location of lncRNA MVIH within the RPS24 gene is shown.Exons and intron of the RPS24 gene are indicated byblack boxes and black lines,and lncRNA MVIH is depicted as the gray box.The location of the forward(F)and reverse(R)primers used for RT-PCR and nested PCR are shown.Orientation of arrows indicates the direction of the transcription or amplification reaction.(C)Linear correlationbetween the expression of the MVIH and RPS24 was not observed.DCt values were used to measure gene expression,which was normalized byb-actin expression levels.(D)Expression of RPS24 was decreased,and MVIH was not significantly changed in SMMC-7721 and HCCLM3 cellswith two different siRNAs against RPS24.Statistical differences were analyzed relative to siRNA control.$P 0.05.HEPATOLOGY,Vol.56,No.6,2012YUAN ET AL.2233lncRNA MVIH expression level in HCC was associ-ated with specific clinicopathological characteristics.We measured MVIH expression level in tumor tissuesfrom another 215 HCC patients independent from 40HCC patients of cohort 1(Supporeing Table 1,cohort2)by qRT-PCR.Median expression level was used asthe cutoff.Low MVIH expression in 107 patients wasclassified as values below the 50th percentile(with anaverage DCt expression value of 6.863,ranking from5.915 to 8.318,when compared with b-actin).HighMVIH expression in 108 patients was classified as val-ues above the 50th percentile(with an average DCtexpression value of 4.149,ranking from 2.249 to5.526,when compared with b-actin).We found that ahigher MVIH expression level was significantly morefrequent in tissues with increased microvessel invasion(v2 6.267;P 0.016)and advanced TNM tumornode metastasis T stage(v2 9.332;P 0.009;Table 1).We further examined whether MVIH expressionlevel correlated with outcome of HCC patients afterhepatectomy.Kaplan-Meiers survival curves were usedto compare the low(n 107)and high(n 108)lncRNA MVIH subgroups,and the results are pre-sented in Fig.2A,B.Remarkably,patients with higherMVIH expression level had poorer RFS(P 0.001)and overall survival(OS;P 0.007).A univariate analysis revealed that the gender,alpha-fetoprotein(AFP),tumor size,Edmondsons grade,mi-crovascular invasion,macrovascular invasion,patholog-ical satellite,encapsulation,TNM stage,BCLC stage,and MVIH expression level were significantly corre-lated with RFS or OS(Supporting Table 3).All 11clinicopathological characteristics were further appliedfor multiple analyses.Coxs multivariate proportionalhazards model indicated that tumor size(hazard ratioHR:2.541,95%confidence interval CI:1.519-4.254;P 0.001),pathological satellite(HR:2.365,95%CI:1.476-3.789;P 55:?5548:5935:730.069Gender,male/female94:1385:230.099HBsAg,positive/negative97:10100:80.632HBeAg,positive,negative31:7637:710.464Liver cirrhosis,with/witout64:4376:320.117Serum albumin(g/L),40:?4077:3082:260.537Serum bilirubin,lmol/L,17:?1723:8434:740.122ALT,U/L,40:?4045:6254:540.275AFP,ug/L,20:?2073:3485:230.091Tumor size,cm,5:?530:7744:640.062No.tumor,solitary/multiple83:2490:180.307Edmondsons grade,III:III35:7234:740.884Microvascular invasion,present/absent23:8440:680.016Macrovascular invasion,present/absent8:9910:980.806Micrometastases,present/absent43:6444:641.000Encapsulation,no/complete51:5643:650.273TNM stage,I:II:III78:16:1357:29:220.009,*BCLC stage,A:B:C83:16:881:17:100.873Data are expressed as ratios.Abbreviations:HBsAg,hepatitis B surface antigen;HBeAg,hepatitis B e anti-gen;ALT,aalanine aminotransferase.*Median expression level was used as the cutoff.Low expression of lncRNAMVIH in 107 patients was classified as values below the 50th percentile.HighlncRNA MVIH expression in 108 patients w