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Novel Insights Into the Role of MicroRNA in Lung Cancer Resistance to Treatment and Targeted

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TumorBiol.DOI10.1007/s13277-015-3627-4RESEARCHARTICLEAberrantexpressionoflongnoncodingRNAsincolorectalcancerwithlivermetastasisLe-chiYe1,2&LiRen2&Jun-junQiu3&De-xiangZhu2&TaoChen2&Wen-juChang2&Shi-xuLv1&JianminXu2Received:#14April2015/Accepted:27May2015InternationalSocietyofOncologyandBioMarkers(ISOBM2015AbstractLongnoncodingRNA(lncRNAplaysacrucialroleintheregulationofvariouscellularprocessesandhumandiseases.However,littleisknownabouttheroleoflncRNAsincolorectallivermetastasis(CLM.Inthepresentstudy,weaimedtodeterminewhetherlncRNAsaredifferentiallyexpressedinCLMtissueandtofurtherassesstheirclinicalvalue.lncRNAarrayswereemployedtoscreenfordifferen-tiallyexpressedlncRNAsincolorectalcancer(CRCtissueswithsynchronous,metachronous,ornonlivermetastasis.Basedonbioinformaticsdata,aquantitativereverse-transcriptionpolymerasechainreaction(qRT-PCRassaywasperformedtoidentifytargetlncRNAsinanexpandedsetofCRCsampleswithvarioussubtypesoflivermetastasis.Li-chiYeandLiRencontributedequallytothiswork.ElectronicsupplementarymaterialTheonlineversionofthisarticle(doi:10.1007/s13277-015-3627-4containssupplementarymaterial,whichisavailabletoauthorizedusers.*JianminXu10198806@qq.comLe-chiYeyljwenzhou@126.comLiRen1993765465@qq.comJun-junQiuyeletian7777@163.comDe-xiangZhu2957037980@qq.comTaoChen905258048@qq.comTherelationshipsbetweenthetargetlncRNAsandtheclinicalcharacteristicsandpatientprognosiswerefurtheranalyzed.AfterdeterminingtheexpressionprofileoflncRNAs(n=1332inCLMtissue,40differentiallyexpressedlncRNAsthatwerepotentiallyrelatedtoCLMwereselectedforfurtherexaminationinanexpandedsetofclinicalsamples,andthreenoveltargetlncRNAs,termedlncRNA-CLMAT1-3,werever-ified.HighlncRNA-CLMAT3expressionstronglycorrelatedwithlivermetastasis(P=0.03andlymphnodemetastasis(P=0.009.Moreover,patientsdisplayinghighlncRNA-CLMAT3expressionexhibitedashortermedianoverallsur-vivaldurationthanthosedisplayinglowlncRNA-CLMAT3expression(30.7vs.35.2months,P=0.007.MultivariateWen-juChang1059647116@qq.comShi-xuLvrenee15@163.com1DepartmentofOncologicalSurgery,TheFirstAffiliatedHospitalofWenzhouMedicalUniversity,Wenzhou325000,People’sRepublicofChina2DepartmentofGeneralSurgery,ZhongshanHospital,FudanUniversity,Shanghai200000,People’sRepublicofChina3DepartmentofGynecology,ObstetricsandGynecologyHospitalofFudanUniversity,Shanghai,People’sRepublicofChina
analysisdemonstratedthatthelncRNA-CLMAT3expressionlevelisanindependentprognosticfactor(hazardratio2.05,P=0.02afteradjustingforotherknownprognosticfactors.lncRNA-CLMAT3over-expressionwassignificantlyassoci-atedwithCLMandwasanindependentpredictorofpoorsurvivalforpatientswithCRC.Keywords.Colorectallivermetastasis.LongnoncodingRNAlncRNA-CLMAT3BackgroundColorectalcancer(CRC,whichisthesecondmostcommoncauseofcancer-relateddeathworldwide,placesamajoreco-nomicburdenonglobalhealthcaresystems[1,2].Theliveristhemostfrequentsiteofmetastasesfromcolorectalcancer,andapproximately50%ofpatientswithadvancedCRCal-readysufferfromorwilldevelopcolorectallivermetastasis(CLMduringthediseasecourse[3].Intheclinic,althoughCRCpatientsinitiallylackinglivermetastasesundergoR0resectionoftheprimarytumor,someofthesepatientsultimatelydevelopCLM[4],whereasotherpatientsneverexperienceCLM,evenifallpatientsreceivethesamepathologicstagingandtherapy[5].Therefore,additionalprognosticmarkersareurgentlyneededtoindicatethepoten-tialforCLMatthetimeofdiagnosisandtoguidetherapeuticdecisionsinadjuvantsettingsduringeventheearlystageofCRC.Additionally,thediagnosisofandthetherapeuticstrat-egiesforCRCrelapseandmetastasisareprimarilybasedonthetumor-node-metastasis(TNMstagingcriteria[6].How-ever,thismethodconsistentlyresultsinmisdiagnosesbecauseitignoresthemolecularmechanismsunderlyingcancerpro-gression[7].Inrecentyears,advancesingenome-wideanalysesoftheeukaryotictranscriptomehaverevealedthatmostofthehu-mangenomeispervasivelytranscribed,producingalargequantityoflongnoncodingRNAs(lncRNAsmappedtointronicandintergenicregions[8].ThelengthoflncRNAsrangesfromapproximately200nttogreaterthan100kb[8].AsanewlydiscoveredtypeofregulatoryRNAmolecule,lncRNAsmayplayrolesinregulatinggeneexpressioninvar-iousprocessesofepigenetics,transcription,posttranscription,andtranslationduringthedevelopmentofcancer[9].Increas-ingevidencehasindicatedthatlncRNAsplayimportantrolesinCRC[10],suchasHOXtranscriptantisenseRNA(HOTAIR[11],highlyupregulatedinlivercancer(HULC[12],andH19(imprintedmaternallyexpressedtranscript[13].LncRNAswerereportedtobeabnormallyexpressedinCRCtissuesandtobeassociatedwithcellproliferation,apoptosis,cellcycledistribution,andmetastasis[14,15].Forinstance,CRC-associatedtranscript1(CCAT1wasreportedtobehighlyexpressedinCRC,especiallyinmetastatictissue[16];lowTumorBiol.lncRNA-LOC285194expressionwasshowntosignificantlycorrelatewithdistantmetastasesinpatientswithCRC[17].However,nomapoftheextensiveandchoreographedtran-scriptionoflncRNAsinCRCorCLMhasbeengenerated.Moreover,patientswithmetastaticCRCwereroughlystratifiedintoCLMandnon-CLMsubgroupsintraditionalstudieswith-outfurthersubdividingtheCLMpatientpopulation.Inthisstudy,weprofiledthelncRNAexpressionlevelsofCRCsampleswithvariouslivermetastasesusingahumanlncRNAarray.ThedifferentiallyexpressedlncRNAswereiden-tifiedbasedoncomparisonsbetweendifferenttissues.Inaddi-tion,aquantitativereverse-transcriptionpolymerasechainreac-tion(qRT-PCRassaywasperformedtoidentifytargetlncRNAsinexpandedCRCsampleswithvariouslivermetastases.MethodsPatientsamplesandstudydesignThepatientswereeligibleforinclusioniftheirprimarytumorshadbeenresectedandiftheysufferedfromhistologicallyconfirmedcolorectaladenocarcinomabasedonthepTNMclassificationasstageIIIorIVaccordingtotheNationalCom-prehensiveCancerNetworkguidelines.Othereligibilitycriteriawereage>18and<75yearsandRNAextractedfromeachsampledisplayingnoevidenceofdegradationorDNAcontamination.Thepatientswereexcludediftheyhadprevi-ouslybeenexposedtoanytargetedtherapy,chemotherapy,radiotherapy,orinterventiontherapyforCRC.The264tissuesamplesfromthepatientswhomettheeli-gibilitycriteriawerecollectedattheZhongshanHospitalofFudanUniversitybetweenJanuary2009andJuly2012.Thisstudyhasbeenapprovedbythelocalethicscommittee.Allsampleswerefrozeninliquidnitrogen,storedat−80°C,andallocatedintothreephasesoffurtheranalysisinchronologicalorder(Fig.1.(1Discoveryphase.Atotalof18CRCtissuesand6normalcolorectaltissueswereusedforlncRNAmicroarrayanal-ysis.Moreover,thetumortissueswereequallycatego-rizedintothreegroups[18]:nonlivermetastasis(NLM,definedasnolivermetastasesoccurringforatleast2yearsaftertheprimarydiagnosisofCRC,synchro-nouslivermetastasis(SLM,definedaslivermetastasesoccurringwithin6monthsoftheprimarydiagnosisofCRC,andmetachronouslivermetastasis(MLM,de-finedaslivermetastasesoccurringbeyond6monthsaftertheprimarydiagnosisofCRC.DiffGeneanalysis(ttestandsignificanceanalysisofmicroarrayswasperformedtoidentifythedifferentiallyexpressedlncRNAsbasedonfourpairwisecomparisons(theformerdefinedastheexperimentalarmandthelaterdefinedasthecontrol
TumorBiol.Fig.1Thestudydesign.CLMcolorectallivermetastasis,CRCcolorectalcancer,NCRnormalcolorectum,NLMCRCwithnolivermetastasis,MLMCRCwithmetachronouslivermetastasis,SLMCRCwithsynchronouslivermetastasis,qRT-PCRquantitativereverse-transcriptionpolymerasechainreaction,andlncRNAlongnoncodingRNAarm:SLMversusNLM,MLMversusNLM,SLMver-susMLM,andCRCtissueversuspairednormaltissuefromSLM.ThedifferentiallyexpressedlncRNAsthatdisplayeda>1.5-foldchangeinexpressionandaPvalue<0.01basedonatleasttwopairwisecomparisonswiththesametrendwereselectedforfurtherexaminationinthetrainingphase.(2Trainingphase.Atotalof40lncRNAsidentifiedviamicroarraywereinitiallyanalyzedviaqRT-PCRinanindependentcohortofpairedtumorandnormaltissuesamplesfrom30patients(10NLM,10SLM,and10MLM.OnlythelncRNAsdisplayingfoldchangesinexpressionthatcorrespondedtothemicroarrayresultswereselectedforfurtheranalysisinthevalidationphase.(3Validationphase.AtotalofthreenovellncRNAs(termedlncRNA-CLMAT1-3wereidentifiedfromthetrainingphase,andlncRNA-CLMAT3wasselectedforfurtheranalysisinanindependentcohortofpairedtumorandnormaltissuesamplesfrom90CRCpatientswithorwithoutlivermetastases.Theoverallsurvivalduration(OSwascalculatedfromthedateoftheconfirmationoftheCRCdiagnosistothedateofdeathresultingfromanycauseorthedateofthemostrecentfollow-up,atwhichpointthedatawerecensored.(asindicatedbyanRNAintegrityvalue≥7.0anda28S:18Sratio≥0.7wasprocessedforfurtheranalysis.ThelncRNAexpressionprofileswereobtainedusingtheGlueGrantHumanTranscriptomeArray,whichwasmanufacturedbyAffymetrixandStanfordUniversity.Thismicroarraycontainedprobesfor563,097noncodingRNAscovering50,783codinggenes,including4408lncRNAs,col-lectedfromtheRefSeq,EnsemblandUCSCKnownGenesdatabasesbasedonhumangenomeassemblyhg18.Afterwashingtheslides,thearrayswerescannedusingtheGeneChip®Scanner3000.TherawdatawereobtainedusingCommandConsoleSoftware3.1accordingtothedefaultset-tingsandwereprocessedusingAffymetrixPowerToolswithrobustmulti-arrayanalysisforbackgroundcorrection,nor-malization,andsummarization.TheextractedRNAsampleswereusedtosynthesizefirst-strandcDNAusingtheSuperScriptIIIFirst-StrandcDNASynthesisKit(Invitrogen.qRT-PCRanalysiswasperformedusingtheAppliedBiosystems(ABIPrism7900Real-TimePCRsystem(ABI,USA,FastStartUniversalSYBRGreenMasterMix(Roche,andtheprimerspresentedinSupplemen-talTableS1.ThenormalizedrelativegeneexpressionlevelswerecalculatedaccordingtothestandardΔΔCtmethodusingABIRQManagerSoftware(v1.2.EachqRT-PCRre-actionwasrepeatedthreeseparatetimes,whichincludedtech-nicaltriplicatesineachreaction.StatisticalanalysesThedatawereexpressedasthemeans±standarderrorofthemean.Alloftheenumerateddatawerecomparedusingthechi-squaretest,andcomparisonsofthecontinuousdatabe-tweentwogroupswereanalyzedusinganindependentttest.Forcomparisonsofmorethantwogroups,ANOVAwasused,andfurthercomparisonbetweentwogroupswasperformedlncRNAmicroarrayandqRT-PCRTotalRNAwasextractedfromfrozentissuesusingTRIzolreagent(Invitrogen,Carlsbad,CA,USAaccordingtothemanufacturer’sprotocol.AnAgilent2100Bioanalyzer(AgilentTechnologies,SantaClara,CA,USAwasusedtoquantifytheRNAandtoevaluateitsintegrity.RNAdisplayingnoevidenceofdegradationorDNAcontamination
TumorBiol.usingBonferronianalysis.SurvivorfunctionswereestimatedusingtheKaplan–Meiermethod,includingthelog-ranktestandmultivariateCoxregressionanalysis(Waldforward,forthevariablesthatdisplayedsignificanceatP<0.05basedonunivariateanalysis.SPSSsoftware(version16.0;SPSS,Chi-cago,IL,USAwasusedforthestatisticalanalyses.APvalue<0.05wasconsideredtobesignificant.Discoveryphase(lncRNAscreeningandtestingToconfirmtheaccuracyofthemicroarrayscreen,theexpressionoffiverandomlyselectedlncRNAswasan-alyzedviaqRT-PCR.Theresultsconfirmedthattherel-ativemRNAexpressionlevelswerehighlyconsistentwiththosedemonstratedbymicroarrayanalysis(SupplementalFigureS1.HierarchicalclusteringshowedsystematicvariationsintheexpressionoflncRNAsintheCLMsamples(SupplementalFigureS2.Comparedwiththenormalcolorectaltissues,atotalof270lncRNAsdisplayeddifferentialexpression(P<0.05intumortissuesfromtheSLMpatients,including180upregulatedlncRNAsand90downregulatedlncRNAs.ComparedwiththeNLMgroup,948(790upregulatedand158downregulatedlncRNAsintheSLMgroupand320(273upregulatedand47downregulatedlncRNAsintheMLMgroupweresignificantlydifferentiallyexpressedinthetumortissues.ComparedwiththetumortissuefromtheMLMpatients,466lncRNAs,including359upregulatedResultsPatientcharacteristicsNosignificantdifferencesinthebaselinecharacteristicswereobservedbetweenthethreesubgroups(NLM,SLM,andMLMduringthediscoveryortrainingphase(SupplementalTablesS2andS3..Additionally,nosignificantdifferencesinthedistributionofage,sex,ortumorlocationorsizewereobservedbetweenthetwosubgroupsduringthevalidationphase(Table1.Table1AssociationoflncRNA-CLMAT3withclinic-pathologicalcharacteristicsCharacteristicsNumberCLMAT3expressionaLow(%High(%29(64.416(35.627(60.018(40023(51.122(48.921(40.024(60.028(62.217(37.87(15.638(84.411(24.434(75.622(48.923(51.125(55.620(44.418(41.98(18.617(39.5PvalueGender:Age:Lesionlocation:Tumorsize:Histologicalgrade:Tumorinvasionb:Lymphnodemetastasis:NegativePositiveCEA:Livermetastasis:Therapy(aftersurgeryMaleFemale≦60>60ColonRectum≦5cm>5cmI~IIIII~IVT1~2T3~460305535454542486228187233575040603041212631(68.914(31.128(62.217(37.822(48.923(51.121(53.324(46.733(73.312(26.711(24.434(75.623(51.122(48.928(62.217(37.835(77.810(22.223(51.113(28.99(20.00.660.830.831.000.260.290.0090.200.030.12<5ng/μL≧5ng/μLNegativePositiveCC+RTcombineCEAcarcinoembryonicantigen,Cchemotherapy,Rradiotherapy,TcombinetargettherapycombinedwithchemotherapyorradiotherapyaAccordingtothelncRNA-CLMAT3/GAPDHexpressionratiointhecanceroustissues,90patientswerestrat-ifiedintothehighlncRNA-CLMAT3expressiongroup(n=45orthelowlncRNA-CLMAT3expressiongroup(n=45AccordingtotheNCCNguidelines,T1–2designatesaprimarytumorinvadingthesubmucosaorthemuscularispropria,andT3–4designatesaprimarytumorinvadingbeyondthemuscularispropriab
TumorBiol.lncRNAsand107downregulatedlncRNAs,weresignificant-lydifferentiallyexpressed(P<0.05inthetumortissuefromtheSLMpatients.Furthermore,293lncRNAsdisplayeda>1.5-foldchangeinexpressionandaPvalue<0.01amongthefourpairwisecomparisons;40lncRNAsmetthesecriteriainmorethanonepairwisecomparisonandwereselectedforfurtheranalysisinthetrainingphase.Trainingphase(verificationofthemicroarrayresultsBasedonqRT-PCRanalysisofthese40lncRNAsintheex-pandedclinicalsamples,wefoundthatthefoldchangesintheexpressionofthreelncRNAs(TR140014124,TR01015341,andTR05005298wereconsistentwiththemicroarrayresults(Fig.2.ThemRNAlevelsofallthreelncRNAsweresignif-icantlyupregulatedintheNLMtissuescomparedwiththenormalcolorectaltissuesandweresignificantlyhigherintheSLMtissuesthanintheNLMtissues.Furthermore,bothTR01015341andTR140014124showedhigherexpressionintheSLMsamplesthanintheMLMsamples(P=0.048andP=0.03,respectively,andsignificantdifferencewithinthethreegroups(NLM,MLM,andSLMbasedonone-wayANOVA(P=0.02andP=0.002,respectively.Theabovere-sultsdemonstratedthatTR140014124,TR01015341,andTR05005298wereupregulatedintheCRCtumortissue,im-plyingthatthesethreelncRNAsmayplayaroleinCLMprogression.TheTR140014124,TR01015341,andTR05005298se-quenceswereanalyzedusingtheNCBI(http://www.ncbi.nlm.nih.gov/andUCSC(http://www.genome.ucsc.edu/blastgenomedatabases.Inaddition,arelevantlncRNAdatabasewassearchedforfurtheranalysis.AllofthesesearchesindicatedthatthesethreelncRNAsequenceshavenotbeenofficiallyreportedpreviously(datanotshown.ConsideringthatthesethreelncRNAswereidentifiedforthefirsttimeinCLMtissuesamplesviamicroarrayscreening,wetermedthesegeneslncRNA-CLM-associatedtranscript1–3(CLMAT1-3accordingtothemethodusedtonamepreviousgenes,suchasmetastasis-associatedlungadenocarcinomatranscript1(MALAT-1[19]andCCAT-1[16].Fig.2TherelativeexpressionlevelsofTR140014124,TR01015341,andTR05005298werecomparedbetweentheCRCandnormaltissuesamplesorbetweenCRCtissuesofvaryinglivermetastaticstatusviaqRT-PCR.a,b,eTherelativeexpressionlevelsofTR140014124,TR01015341,andTR05005298,respectively,weresignificantlyhigherinthetumortissuesthaninthenormaltissues.b,d,fThemRNAlevelsofTR140014124,TR01015341,andTR05005298,respectively,intheSLMandMLMtissuesweresignificantlyhigherthanthoseintheNLMtissues.*P<0.05;**P<0.01.CRCcolorectalcancer,NLMCRCwithnolivermetastasis,MLMCRCwithmetachronouslivermetastasis,andSLMCRCwithsynchronouslivermetastasis
TumorBiol.Validationphase(clinicalvalueofthelncRNA-CLMATsWefocusedonlncRNA-CLMAT3,locatedonhumanchro-mosome14(chr14:101379770-101381326,hg19,becauseitwasthemoststronglyupregulatedlncRNAintheCRCtissuescomparedwiththenormaltissues(Fig.2,displayingatumortissue/normaltissueexpressionratioof2.21and2.10intheSLMandMLMgroups,respectively.ToassessthecorrelationoflncRNA-CLMAT3expressionwiththeclinic-pathologicalcharacteristics,lncRNA-CLMAT3expressionin90CRCandnormalcolorectaltissueswasfurtheranalyzedviaqRT-PCR.ThelevelsoflncRNA-CLMAT3inthecanceroustissueswere2.26-foldhigherthanthoseinthenormalcolorectaltissues(P<0.05,Fig.3a.Inaddition,lncRNA-CLMAT3expressionwassignificantlyhigherinthepatientswithlivermetastasisthaninthosewith-outlivermetastasisandinthepatientswithlymphnodeme-tastasisthaninthosewithoutlymphnodemetastasis(Fig.3b,c.AccordingtothelncRNA-CLMAT3/glyceraldehyde3-phosphatedehydrogenase(GAPDHexpressionratiointhecanceroustissues,thesecaseswerestratifiedintoahighlncRNA-CLMAT3expressiongroup(n=45andalowlncRNA-CLMAT3expressiongroup(n=45.WefoundthathighlncRNA-CLMAT3expressionwassignificantlyassoci-atedwithlivermetastasisandlymphnodemetastasis.TheseresultsindicatedthathighlncRNA-CLMAT3expressionisrelatedtoCRCprogression(Table1.Fig.3ThelncRNA-CLMAT3expressionlevelsin90pairedCRCandnormaltissuesampleswereanalyzedviaaqRT-PCRassay.aTheexpressionoflncRNA-CLMAT3wassignificantlyhigherinthetumortissuesthanthatinthenormaltissues.bThelncRNA-CLMAT3expressionlevelsweresignificantlyhigherinthetumorswithlivermetastasisthaninthosewithoutlivermetastasis.cTheexpressionoflncRNA-CLMAT3wassignificantlyhigherinthelymphnodemetastasissamplesthaninsampleswithoutlymphnodemetastasis.dThepatientsdisplayinglowlncRNA-CLMAT3expressionexhibitedlongerOSthanthosedisplayinghighlncRNA-CLMAT3expressions.*P<0.05;**P<0.01.LMlivermetastasisandlncRNA-CLMAT3colorectallivermetastasis-associatedtranscript3WefurtherinvestigatedtheassociationoflncRNA-CLMAT3expressionwithOSinpatientstoevaluateitsprognosticvalue.ThemedianOS(MSTforallpa-tientswas35.2months(95%confidenceinterval31.0~39.4months.ThepatientswithtumorsdisplayinghighlncRNA-CLMAT3expressionexhibitedshorterOSthanthepatientswithtumorsdisplayinglowlncRNA-CLMAT3expression(Fig.3d,MST30.7vs.35.2months,P=0.007.Furthermore,univariateanalysisrevealedthatlivermetastasis(P=0.012andlncRNA-CLMAT3expression(P=0.008wereprognosticindica-torsofdisease-specificsurvival.Afterincludingthevar-iables(i.e.,lncRNA-CLMAT3high/lowexpression,gen-der,age,lesionlocation,tumorsize,histologicalgrade,tumorinvasion,lymphnodemetastasis,CEA,liverme-tastasis,andpostoperativetherapyintheCoxregres-sionmodel,multivariateanalysisshowedthatlncRNA-CLMAT3expression(P=0.02andlivermetastasis(P=0.04wereindependentprognosticindicatorsofsurvivalforpatientswithCRC(Table2.DiscussionAlthoughtherehavebeenmultiplestudiesofthemRNAormicroRNAtranscriptomeinCRCorCLM[20],thisisthefirststudythatdescribestheexpressionprofilesof
TumorBiol.Table2Multivariateanalysisoftheclinic-pathologicalcharacteristicsassociatedwithoverallsurvivaldurationinthevalidationphaseVariableHazardratio95%CIPvalueLivermetastasis(with/without1.911.03~3.560.04LncRNA-CLMAT3expression2.051.10~3.820.02(high/lowhumanlncRNAsinCLMbasedonmicroarrayanalysis.ConsideringthatSLMconfersasignificantlypoorerprognosisthanMLM[18],wefurthersubdividedtheCLMpatientsintoNLM,MLM,andSLMpatients,whichhelpedtoidentifymoremeaningfullncRNAsasbiomarkersforCLM.Additionally,asdemonstratedinthediscoveryphaseofthisstudy,thecomparisonbe-tweentheSLMandMLMgroupsrevealedsignificantlyfewerdifferentiallyexpressedlncRNAsthanthecom-parisonbetweentheSLMandNLMsubgroups(466vs.948,P<0.001.ThisfindingindicatedarelationshipbetweentheMLMandSLMsubgroupsregardingtheprimarytumoratamolecularlevel.IntheexaminationofpotentiallncRNAsasmolecularmarkersforcancer,traditionalstudieshavebeenlimitedtothecellularlevelorhavedependedonisolatedtissueverifica-tion.Thisstudytookfulladvantageofthepreliminarysamplebankandallocatedthetissuesamplestothreephasesinchro-nologicalorder,fromlncRNAscreeningtotargetlncRNAverification,whichpavedthewayfortheanalysisofCLM-associatedlncRNAs.Finally,weareparticularlyinterestedinlncRNA-CLMAT3becauseitsover-expressiondisplaysclin-icalvalue.Toourknowledge,thisisthefirstreportonthedysregulatedexpressionpatternoflncRNA-CLMAT3inCRC.Moreimportantly,wefoundthatthelncRNA-CLMAT3expressionlevelwassignificantlyassociatedwithlivermetastasisandsurvivalamongpatientswithCRC.Spe-cifically,patientswithhighlncRNA-CLMAT3expressionlevelsexhibitedpoorprognosis.lncRNAsmayfunctionastumorsuppressorsandonco-genesincancerinamannerthatisidenticaltoprotein-codinggenes[21,22].Inthepresentstudy,wefoundthatCRCpatientsdisplayinghighexpressionlevelsoflncRNA-CLMAT3morefrequentlydevelopedlivermetastasesthanthosewithlowlncRNA-CLMAT3expressionlevels,indicat-ingthatlncRNA-CLMAT3mayactasanoncogene.EmergingdatastronglydemonstratethatlncRNAspro-motecancerprogressionprimarilybyregulatingprotein-codinggenes[23].Forexample,thedownregulationoflncRNA-MALAT-1attenuatedWnt/β-cateninsignaling,therebyinhibitingCRCinvasionandmetastasis[24];LncRNA-CCAT2,reportedtopromoteCRCcellgrowthandmetastasis,mayregulateMycinCRCpathogenesis[25].Moreover,somelncRNAscouldfunctionviatheregulationofneighboringprotein-codinggenes(e.g.,lncRNA-Evf-2transcribedfromtheultra-conservedDlx-5/6regionfunctionsasatranscriptionalcoactivatorofDlx-2[26].Basedonanal-ysisusingtheUCSCgenomedatabase(hg19,wefurtherfoundthattherewasoneprotein-codinggene,secretedproteinacidicandrichincysteine(SPARC[27],locatedintheanti-sensestrandadjacenttolncRNA-CLMAT3(SupplementalFigureS3.ItwasreportedthatSPARCisover-expressedinhighlymetastatictumors(e.g.,endometrialcancer,melanoma,glioblastomas,prostatecancer,andbreastcancerandpro-motestumormetastasis[27–29].Thus,incombinationwiththeresultsofourstudy,thesefindingsledtoourhypothesisthatlncRNA-CLMAT3mayregulateSPARCexpressionatthelevelsoftranscription,chromatinmodification,andpost-transcriptionalprocessing,toparticipateinCRCprogressionormetastasis.FutureexperimentsarerequiredtoelucidatetheexactmechanismsbywhichlncRNA-CLMAT3affectsCLMbyregulatingSPARCexpression.Ourstudycontainsotherlimitations.First,thenumberoftissuesanalyzedwaslimited(especiallyfortheMLMsub-group,andthefollow-updurationwasshort.Therefore,theresultsofcertainsubgroupanalysesareunconfirmed,andthe5-yearoverallsurvivalratehasyettobedetermined.Second,alltissuesampleswerecollectedfromonetreatmentcenterandthecorrespondingpatientswerelimitedtotheHannation-ality,whichmighthaveresultedinaminimalstudybiasbe-causeofthepotentiallydistinctexpressionoflncRNAsintissuefromindividualsofdifferentnationalities.FurtherinvitroandinvivoexperimentsarecurrentlybeingperformedbyourgrouptoinvestigatetheeffectoflncRNA-CLMAT3onthelivermetastasisofCRC.ConclusionsWeidentifiedonenoveldysregulatedlncRNA,termedCLMAT3,intissuesamplesfromvariousCLMsubtypesbasedonamicroarrayscreenandstep-by-stepqRT-PCRverification.OurstudydemonstratedthathighexpressionoflncRNA-CLMAT3wassignificantlyassociatedwithlivermetastasisofCRCandwasanindependentprognosticindicatorofsur-vivalforpatientswithCRC.CCAT-1,coloncancer-associatedtranscript-1;CLM,colo-rectallivermetastasis;CLMAT3,colorectallivermetastasis-associatedtranscript3;CRC,colorectalcancer;HULC,highlyupregulatedinlivercancer;LncRNA,longnoncodingRNA;MALAT-1,metastasis-associatedlungadenocarcinomatran-script1;MLM,metachronouslivermetastasis;NLM,non-livermetastasis;qRT-PCR,quantitativereverse-transcriptionpolymerasechainreaction;SLM,synchronouslivermetasta-sis;SPARC,secretedproteinacidicandrichincysteine;TNM,tumor-node-metastasis
AcknowledgmentsWethanktheNationalNaturalScienceFoun-dationofChina(No.81372315,Shanghaimedicalguideproject(134119a4800andShanghaiScientificResearchPlanProject(No.13JC1401601forprovidingfundingsupports.ConflictsofinterestNoneAuthors’contributionsLe-chiYeandLiRenperformedthemoleculargeneticsexperiments,participatedinthesequencealignment,anddraftedthemanuscript.Jun-junQiuparticipatedinthesequencealignment.TaoChen,De-xiangZhu,andWen-juChangparticipatedinthedesignofthestudyandperformedthestatisticalanalysis.JianminXu,LiRen,andYeWeiconceivedofthestudy,participatedinitsdesignandcoordination,andhelpedtodraftthemanuscript.Allauthorsreadandapprovedthefinalmanuscript.References1.SiegelR,DesantisC,JemalA.Colorectalcancerstatistics,2014.CACancerJClin.2014;64:104–17.2.BrennerH,KloorM,PoxCP.Colorectalcancer.Lancet.2014;383:1490–502.3.SteeleJrG,RavikumarTS.Resectionofhepaticmetastasesfromcolorectalcancer.Biologicperspective.AnnSurg.1989;210:127–38.4.ChuangSC,SuYC,LuCY,HsuHT,SunLC,ShihYL,etal.Riskfactorsforthedevelopmentofmetachronouslivermetastasisincolorectalcancerpatientsaftercurativeresection.WorldJSurg.2011;35:424–9.5.MekenkampLJ,KoopmanM,TeerenstraS,vanKriekenJH,MolL,NagtegaalID,etal.Clinicopathologicalfeaturesandoutcomeinadvancedcolorectalcancerpatientswithsynchronousvsmetachronousmetastases.BrJCancer.2010;103:159–64.6.HayashiN,ItoI,YanagisawaA,KatoY,NakamoriS,ImaokaS,etal.Geneticdiagnosisoflymph-nodemetastasisincolorectalcan-cer.Lancet.1995;345:1257–9.7.ManfrediS,LepageC,HatemC,CoatmeurO,FaivreJ,BouvierAM.Epidemiologyandmanagementoflivermetastasesfromco-lorectalcancer.AnnSurg.2006;244:254–9.8.PontingCP,OliverPL,ReikW.EvolutionandfunctionsoflongnoncodingRNAs.Cell.2009;136:629–41.9.MercerTR,MattickJS.StructureandfunctionoflongnoncodingRNAsinepigeneticregulation.NatStructMolBiol.2013;20:300–7.10.XueY,MaG,GuD,ZhuL,HuaQ,DuM,etal.Genome-wideanalysisoflongnoncodingRNAsignatureinhumancolorectalcancer.Gene.2015;556:227–34.11.KogoR,ShimamuraT,MimoriK,KawaharaK,ImotoS,SudoT,etal.LongnoncodingRNAHOTAIRregulatespolycomb-dependentchromatinmodificationandisassociatedwithpoorprog-nosisincolorectalcancers.CancerRes.2011;71:6320–6.12.MatoukIJ,AbbasiI,HochbergA,GalunE,DweikH,AkkawiM.HighlyupregulatedinlivercancernoncodingRNAisoverexpressedinhepaticcolorectalmetastasis.EurJGastroenterolHepatol.2009;21:688–92.13.SorinV,OhanaP,MizrahiA,MatoukI,BirmanT,HochbergA,etal.RegionaltherapywithDTA-H19vectorsuppressesgrowthofTumorBiol.colonadenocarcinomametastasesintheratliver.IntJOncol.2011;39:1407–12.14.ZhengHT,ShiDB,WangYW,LiXX,XuY,TripathiP,etal.HighexpressionoflncRNAMALAT1suggestsabiomarkerofpoorprognosisincolorectalcancer.IntJClinExpPathol.2014;7:3174–81.15.ChenH,XuJ,HongJ,TangR,ZhangX,FangJY.LongnoncodingRNAprofilesidentifyfivedistinctmolecularsubtypesofcolorectalcancerwithclinicalrelevance.MolOncol.2014;8:1393–403.16.NissanA,StojadinovicA,Mitrani-RosenbaumS,HalleD,GrinbaumR,RoistacherM,etal.Coloncancerassociatedtran-script-1:anovelRNAexpressedinmalignantandpre-malignanthumantissues.IntJCancer.2012;130:1598–606.17.LiuQ,HuangJ,ZhouN,ZhangZ,ZhangA,LuZ,etal.LncRNAloc285194isap53-regulatedtumorsuppressor.NucleicAcidsRes.2013;41:4976–87.18.DexiangZ,LiR,YeW,HaifuW,YunshiZ,QinghaiY,etal.Outcomeofpatientswithcolorectallivermetastasis:analysisof1,613consecutivecases.AnnSurgOncol.2012;19:2860–8.19.GutschnerT,HammerleM,EissmannM,HsuJ,KimY,HungG,etal.ThenoncodingRNAMALAT1isacriticalregulatorofthemetastasisphenotypeoflungcancercells.CancerRes.2013;73:1180–9.20.ZorattoF,RossiL,VerricoM,PapaA,BassoE,ZulloA,etal.Focusongeneticandepigeneticeventsofcolorectalcancerpatho-genesis:implicationsformoleculardiagnosis.TumourBiol.2014;35:6195–206.21.NaganoT,FraserP.No-nonsensefunctionsforlongnoncodingRNAs.Cell.2011;145:178–81.22.WiluszJE,SunwooH,SpectorDL.LongnoncodingRNAs:func-tionalsurprisesfromtheRNAworld.GenesDev.2009;23:1494–504.23.WangKC,ChangHY.MolecularmechanismsoflongnoncodingRNAs.MolCell.2011;43:904–14.24.JiQ,LiuX,FuX,ZhangL,SuiH,ZhouL,etal.ResveratrolinhibitsinvasionandmetastasisofcolorectalcancercellsviaMALAT1mediatedWnt/beta-cateninsignalpathway.PLoSOne.2013;8:e78700.25.LingH,SpizzoR,AtlasiY,NicolosoM,ShimizuM,RedisRS,etal.CCAT2,anovelnoncodingRNAmappingto8q24,underliesmetastaticprogressionandchromosomalinstabilityincoloncancer.GenomeRes.2013;23:1446–61.26.FengJ,BiC,ClarkBS,MadyR,ShahP,KohtzJD.TheEvf-2noncodingRNAistranscribedfromtheDlx-5/6ultraconservedregionandfunctionsasaDlx-2transcriptionalcoactivator.GenesDev.2006;20:1470–84.27.YoshimuraT,NagaharaM,KuoC,TurnerRR,Soon-ShiongP,HoonDS.Lymphovascularinvasionofcolorectalcanceriscorre-latedtoSPARCexpressioninthetumorstromalmicroenvironment.Epigenetics.2011;6:1001–11.28.HeitzerE,ArtlM,FilipitsM,ReselM,GrafR,WeissenbacherB,etal.DifferentialsurvivaltrendsofstageIIcolorectalcancerpa-tientsrelatetopromotermethylationstatusofPCDH10,SPARC,andUCHL1.ModPathol.2014;27:906–15.29.YusufN,InagakiT,KusunokiS,OkabeH,YamadaI,MatsumotoA,etal.SPARCwasoverexpressedinhumanendometrialcancerstem-likecellsandpromotedmigrationact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