王紅陽-血清miRNA作為肝細(xì)胞癌的預(yù)測和預(yù)防生物標(biāo)志物

SerummiRNAasPredictive&PreventiveBiomarkerinHepatocellularCarcinomaHong-YangWang王紅陽NationalCenterforLiverCancer,ChinaEasternHepatobiliarySurgeryHospitalShanghaihywangk@WHO(2015)

:782,000newlivercancercasesoccuredintheworld.50%ofnewlivercancercasesinChina,SecondcauseofcancerdeathinChina.HCCandICCarethetwomajorformsofprimarylivercancers.

WHO,Globocan2012ChinaCancerCenter2012LivercancerisasignificantcauseofcancermorbidityandmortalityworldwideEasternHepatobiliarySurgeryHospitalTheNumberofHepatectomyfrom1960-2014Theetiologiesvarygeographically.Chronicinflammationhasbeenidentifiedinmorethan80%ofthecasesandappearstobeakeymediatorinalteringthelivermicroenvironment,increasingtheriskofcarcinogenesis.Themajorityofcasesoccurcirrhosis,HCCasaconsequenceofinflammation.

MultistepprocessofhepatocellularcarcinomaTheHeterogeneityAttributionofCirrhosisandHCCduetoInfectionwithHBVandHCV,inDifferentRegionsAmerican5%Cirrhosis16%HCCEurope13%Cirrhosis18%HCCChina57%Cirrhosis65%HCCAmerican42%Cirrhosis48%HCCEurope38%Cirrhosis44%HCCChina21%Cirrhosis18%HCCJ.Hepatology2006JClin

Oncol2011NatureReviewsCa.2013Difficulttopredict&detectearlyHCCResistanttoconventionaltherapyHighrecurrenceaftersurgicalresectionLackingwell-definedmoleculartargetsandmedicines

Poor

Long-termSurvivalLackingidealbiomarkers—Prediction&EarlyDiagnosisLackingwell-definedtargets—noveldrugsDifficultiesinHCCPrediction,Diagnosis&TreatmentScreeningforBiomarkersofHCC--Prediction,Diagnosis&TreatmentProteomicsMetabolomicsEpigeneticsOurProjectsGenomicsMethylationScreening1p36.3314q32.31chrchr1start1085469end1096038probe_num50hyper_654130.999903778hypo_654139.62E-05hyper_687780.999996386hypo_687783.61E-06hyper_688200.999999996hypo_688203.79E-09hyper_692460.999999721hypo_692462.79E-07hyper_695640.999999981hypo_695641.91679E-08hyper_699210.999779678hypo_699210.000220322hyper_699960.999988668hypo_699961.13317E-05hyper_700421hypo_700423.92418E-18hyper_701881hypo_701885.23287E-22hyper_790050.993551737hypo_790050.006448263Genome-wideMethylationScreening（450Kchips）Genome-widemethylationscreeningwithHCCtissuesandtheircounterpartnormaltissuesTwosignificantlydemethylatedregionsinover70percentageofHCCgenomcis,namedaschromosome1parm36.33andchromosome14qarm32.31miR-429clusterlocatedin1p36.33regionchr1:1,090,000-1,092,500bisulfite

sangersequencing50pairesofHCCsampleswerefurtherexaminedandfourCpGislandsnamedasCpG8,9,10,11upstreamofmir-429promotorwereidentifieddemethylatedintumortissues.IntracellularExpressionofmiR-429OvertenHCCcelllineshumanembryonickidneycell293Thumanmonocytic

leukaemiacellsTHP-1humanlymphoblastcellsU937Negativecorrelationoftheexpressionofmir-429withpromotor

methylationlevel.EnrichmentofmiR-429inHCCComparetheexpressionlevelPresentedstainingofmir-429in242HCCtissuesbyInsituhybridyzationmiR-429isaprognosticfactorofHCCOverallsurvivalDiseasesfreesurvival(paraffin-embeddedHCCtissuearray)SurvivalanalysisshowedthatmiR-429expressionlevelswerenegativelycorrelatedwithoverallsurvivalanddisease-freesurvival.242HCCs,8yearsfollowupClinicopathologicalCharacteristicsof242HCCsmetastasis?development?CancerStemcell?QuestionsmiR-429inHCCHowitworksFunctionsClinicalApplication?Tumor-InitiatingCells(T-ICs)ALDH1MMP2P-AKTOCT4β-CateninNLKC-SRCP-Stat3MMP2MMP9MMP12FIF16ABCBSBMI-1INK4ANANOGHNF4aBCL2PTENCytoplasmNucelusIdentifiedHCCT-ICBiomarkersMulti-BiomarkersComplexSignalPathwayMutli-BiomakersOne-CellVSVSGastroenterology2009NovelliverT-ICsmarker:EpCAM(in

cooperation

with

XinweiWang,NIH)

HeterogeneousExpressionofEpCAM+HCCCellsHPCHCC(EpCAM+AFP+)representsasubsetofinvasiveHCCswithCSCfeatures.SpheroidXenograftsMiR-429isEnrichedinEpCAM+LiverT-ICsLiverTICsmarkers:EpCAM,CD133miR-429enrichedinsortedprimaryEpCAM+orCD133+HCCinitiatingcellsmiR-429increasedtheproportionofEpCAM+liverTICs

butnotCD133+onesExogenousexpressionofmir-429inducedexpansionofTICcellsinvitroandinvivoLimitingdilutionassaySelf-renewalMir-429EnhancesCellProliferationMir-429ProtectsCellfromApoptosisChemoresistancemiR-429promotesthepropertiesofEpCAM+liverT-ICsmiR-429QuestionsmiR-429inHCCHowitworks?FunctionsClinicalApplicationRBBP4isthedirectgeneofmiR-429Negativecorrelation15celllines；161HCCRBBP4(alsoknownasRbAp48,NURF55)involvedinhistone

acetylationandchromatinassemblypartofcorepressorcomplexesbindstoretinoblastomaproteintoregulatecellproliferationinvolvedintranscriptionalrepressionofE2F-responsivegenesThephysiologicalfunctionofRBBP4inHCCTheexpressionofRbbp4wassignificantlydecreasedinHCC.(n=161)KnockdownexpressionofRbbp4leadstotheprogressionofHCCcellsinvivo.ThedistributionofRBBP4inEpCAM-/+HCCsIncreasedlevelofRbbp4inEpCAMnegativeHCCcellsAttachmentSpheroidReattachmentTICsenrichmentassaymir-429-regulatedRBBP4signalisnecessaryfortheexpansionofTICsMir-429TICsExpansion2.65%

→

40.97%RBBP4TheadministrationofRBBP4couldcompletelyblockthemir-429-inducedTICcellsexpansion.Mir-429regulateE2F1activityviaRBBP4RBBP4isinvolvedintranscriptionalrepressionofE2Factivity.E2FActivityReporterAssayTheproteinexpressionofE2F1response-genesOct4expressionismanipulatedbymi429/RBBP4/E2FMiR-429couldbepackedinexosomes

andreleasedbyHCCcellsmiR-429intheintracellularcompartmentwaspositivelycorrelatedwithitslevelinMVs(especiallyexosomes)miR-429wassecretedfromhigh-expressingHCCcellsandrelocatedintothesurroundedtargetcellsviaexosomeIntercellularCommunicationviaExosometheproportionofEPCAM+cells,chemotherapyresistanceability,cellproliferationandtumourspheroidformationweremarkedlyinducedbymiR-429-containingexosomesScience.2014;346(6216):1459-60.QuestionsmiR-429inHCCHowitworksFunctionsClinicalApplication?1.SpecificityofAFPforHCCis76%（viralhepatitis、livercirrhosis、benigndiffuseliverdisease），sensitivityis39-64%.2.Ultrasoundishardtodistinctcirrohosiswithearlyneoplasialesion.SerumbiomarkersforveryearlystageHCCHBsAg+AFP-Ultrasoundimaging-PotentialBiomarker+/?Thegraduallyincreasedmir-429inseruminrodentHCCInden-treatedratlivercancer,serummir-429graduallyincreased.pre-neoplasiaregionInDEN-treatedratliver,mir-429wasfoundenrichedinpre-neoplasiaregion!HGDNistheprecancerouslesionofHCC.Morethan60%ofHGDNstransformedintoHCCduring5years.HGDNLGDNFouryearsSevenCenters9287HBV+195

HCCAlargecohortstudyincluding81,000personsinsevencentersTrainingValidationDiscoveryThreeindependentgroupswereinvolvedindiscovery,training&validationprocesses.NCI’searlydetectionresearchnetworkdefinedphase3studyAbiomarkersetcontainingfivemiRNAswasappliedtopredicttheoccurrenceofHCCbeyondtraditionalmethods.****AscomparedwithAFP,miRNAsetachievedmuchhighersensitivityandspecificity6monthsbeforeultrasounddiagnosis.26%80.8%CancerStemCellTumorBiomarkerTumorProliferationSerummiRNAsincludingmir-429couldbeusedaspredictivebiomarkerforearlydiagnosisandprevention.

GUT2015,2016;Gastroenterology2016……OngoingprospectivestudyConclusionsHCCisaheterogeneousmalignantdisease.Duringtheinitiationtheenvironmentalandgenetic“hits”precipitateasmallnumberoflivercellstoexpressahigherlevelofmiR-429.Theup-regulationofintracellularmiR-429enablesthetransformationofthissetoflivercellstoT-ICsandpromotescellexpansion.ThereleasedmiR-429canentersurroundingnormalcellsthroughMV-mediatedintercellularexchange,whichprovidesapermissivenicheforcellproliferation,self-renewalandtumorigenecity.GroupofbiomarkersincludingmiRNA429forpredictionanddiagnosisareimportanttorealizethepersonalizedmedicine.NationalCentreforLiverCancerThankyouProf.LeiChenDr.LiangLiDr.JingTangDr.JingFu……SurvivalanalysisshowedthatmiR-429expressionlevelswerenegativelycorrelatedwithoverallsurvivalanddisease-freesurvival.miR-429washeterogeneouslyexpressedinHCCsLowmiR-429HighmiR-429LiverTICsmarkers:EpCAM,CD133miR-429enrichedinsortedprimaryEpCAM+orCD133+livercancercellsmiR-429increasedtheproportionofEpCAM+liverTICsbutnotCD133+onesmiR-429enrichedinEpCAM+liverT-ICsmiR-429promotesthepropertiesofEpCAM+liverT-ICsSelf-renewalMalignantproliferationChemotherapeuticresistanceTumorigenicityRBBP4isadirecttargetgeneofmiR-429mRNAexpressionprofiles+softwarepredictionsmiR-429canbesecretedfromHCCcellsandpackedinexosomesmiR-429intheintracellu