小分子RNA與心血管疾病課件

MicroRNAsinVascularDisease

thebeginningofanewtaleChunxiangZhangM.D.,Ph.D.UniversityofMedicineandDentistryofNewJersey.MicroRNAsinVascularDiseaseMiroRNAsMicroRNAs(miRNAs)areanovelclassofendogenous,small,noncodingRNAsthatregulategeneexpression.MaturemiRNAsare18-to24-nucleotide(nt)-long.TheynegativelyregulateproteinexpressionoftheirtargetmRNAsbyeithertranslationinhibitionordegradation.Asagroup,miRNAsareestimatedtoregulateatleast30%ofthehumangenes..MiroRNAsMicroRNAs(miRNAs)PathwayofGeneticInformationFlowandRNA“revolution”

1.Thestandardpathwayofinformationflowinacell.

2.DNATranscriptionandRNAproducts3.ThefirstRNArevolution..PathwayofGeneticInformation

DiscoveryofmiRNA-ThesecondRNA“revolution”

DiscoveryofRNAi.WiththehelpofRNAitechnology,tworegulatorynoncodingRNAswerefound,interferingRNAs(siRNA)andendogenousmiRNAs.TherecentdiscoveriesofRNAiandmiRNArepresentthesecondRNArevolution(Kongetal.GenomicsProteomicsBioinformatics.2005;3:62-72)..

DiscoveryofmiRNA-ThesecomiRNAsandsiRNAs

siRNAsandmiRNAshavesimilarmechanismforgeneexpressionregulation.However,theyaredifferentfromeachother.Thechiefdifferenceliesintheirorigins:SiRNAsareproducedfromlongdouble-stranded(bimolecular)RNAsorlonghairpins,oftenofexogenousorigin.Incontrast,miRNAsareendogenous.Theyareencodedwithinthegenomeandcomefromendogenousshorthairpinprecursor.Therefore,miRNAsaremoreimportantbecausetheyareendogenousregulatorsforgeneexpression..miRNAsandsiRNAssiRNAsandmBiogenesisofmiRNAsandtheirmolecularmechanismforgeneregulation.BiogenesisofmiRNAsandtheirBiologicalfunctionsofmiRNAsAlthoughonlyasmallnumberfromhundredsofidentifiedmiRNAshavebeencharacterized,agrowingbodyofexcitingevidencesuggeststhatmiRNAsareimportantregulatorsforcellgrowth,differentiation,andapoptosis.Therefore,miRNAscouldbetheimportantendogenousregulatorsinnormaldevelopment,physiology,aswellasindisease..BiologicalfunctionsofmiRNAsmiRNAsindiseasesConsequently,dysregulationofmiRNAfunctionmayleadtohumandiseases.Inthisrespect,themostexcitingresearchareaistheroleofmiRNAsincancer,becausecelldedifferentiation,growth,andapoptosisareimportantcellulareventsinthedevelopmentofcancer.Indeed,bothbasicandclinicalstudieshavedemonstratedthatmiRNAsareaberrantlyexpressedindiversecancers.TherecentadvancesintheresearchofmiRNAsandcancershaveresultedinthefollowingconclusions.First,theaberrantexpressionsofmiRNAsaretissue-andcancer-specific.DifferenttissuesandcancershavedifferentmiRNAexpressionprofiles.Second,multiplemiRNAsaredysregulatedincancers.Third,miRNAsarethoughttofunctionasbothtumorsuppressorsandoncogenes.Fourth,miRNAsmayserveasnoveltherapeutictargetsforcancer.Therefore,identifyingthedetailedfunctionsofthekeyaberrantlyexpressedmiRNAsiscriticalforcancerresearch

.miRNAsindiseasesConsequMiRNAsincardiovasculardiseaseProliferativevasculardiseasehaslongbeentheleadingcauseofdeathindevelopedcountries.AlthoughmiRNAsarehighlyexpressedinthevascularsystem,therolesofthesemiRNAsinvasculardiseaseareunknown.Asvasculardiseasesharesomesimilarcellulareventsandmolecularmechanismswithcancer,wehypothesizethatmiRNAsmayplayimportantrolesinvasculardisease.

.MiRNAsincardiovasculardiseaAnimalModelSelectionVascularneointimallesionformation.

Neointimalformationisthecommonpathologicallesioninatherosclerosis,restenosis,diabeticvascularcomplicationandtransplantvasculardisease.

.AnimalModelSelectionVasculHypothesis

Cellgrowth(proliferation)andapoptosisarekeycellulareventsintheformationofvascularneointimallesionformation,whereasmiRNAsareabletoregulatethesecellulareventsinothercelltype.WethereforehypothesizethatmiRNAsmayplayimportantrolesinvascularneointimallesionformationbyregulatingvascularsmoothmusclecellgrowthand/orapoptosis..HypothesisCellgrowth(pMiRNAsinvascularneointimallesionformation

WearetryingtoanswerthefollowingquestionstodeterminethepotentialroleofmiRNAsinvascularneointimalformation.Question#1:ArethemiRNAsaberrantlyexpressedinvascularwallwithneointimallesionformation?

Question#2:Iftheanswertothefirstquestionisyes,dotheseaberrantlyexpressedmiRNAsplayaroleneointimallesionformation?

Question#3:WhatarethecellularmechanismsresponsibleformiRNA-mediatedeffectonneoinimalformatiom?Question#4:WhatarethemolecularmechanismsresponsibleformiRNA-mediatedeffectonneoinimalformatiom?

.MiRNAsinvascularneointimalRatcarotidarteryballooninjuryTodeterminetheexpressionchangesofmiRNAsinthevascularwallwithneointimalformation,weappliedthewell-establishedratcarotidarteryballooninjurymodelinmylaboratory.Inwhich,rat(Sprague-Dawley,250-300g)rightcommoncarotidarteryinjurywasinducedviaaA2FFogartyballooncatheter.Remarkableneointimallesionformationwillbeinducedasearlyas7daysafterinjury..RatcarotidarteryballooninjMiRNAexpressionsignatureinnormalratcarotidartery

.MiRNAexpressionsignatureinMiRNAsareaberrantlyexpressedinratcarotidarteriesafterangioplasty

Comparedwithnormaluninjuredarteries,microarrayanalysisdemonstratedthataberrantmiRNAexpressionwasaremarkablecharacteristicinvascularwallsafterangioplasty.Sevendaysafterballooninjury,113ofthe140arterymiRNAsweredifferentiallyexpressedwithp-value<0.01;60miRNAswereupregulated,and53miRNAsweredownregulated.At14daysafterinjury,110ofthe140arterymiRNAsweredifferentiallyexpressed(63upand47down),while102ofthe140arterymiRNAsweredifferentiallyexpressed(55upand47down)at28daysafterangioplasty.

.MiRNAsareaberrantlyexpresse

ThetimecoursechangesofmiRNAsthatwerehighlyexpressedinratcarotidarteryandover1-foldupregulatedafterangioplasty.ThetimecoursechangesofmiThetimecoursechangesofmiRNAsthatwerehighlyexpressedinratcarotidarteryandover50%downregulatedafterangioplasty.ThetimecoursechangesofmiRConfirmationoftheaberrantmiRNAexpressionininjuredarteriesbyqRT-PCRand/orNorthernblotanalysis.

.ConfirmationoftheaberrantmDotheseaberrantlyexpressedmiRNAsplayaroleinvascularneointimallesionformation?TodeterminethepotentialroleoftheseaberrantlyexpressedmiRNAsinneointimalformation,weselectedmiR-21asourfirstexperimentaltargetforthefollowingreasons:miR-21isoneofthemostup-regulatedmiRNAsafterangioplasty.miR-21isalsoup-regulatedindiversecancers.Thereisacommerciallyavailableinhibitor.

.DotheseaberrantlyexpressedmiR-21inhibitor

TheantisenseoligonucleotideformiR-21ismodifiedateachnucleotidebyanO-methylmoietyatthe2'-riboseposition.Themodifiedantisenseoligonucleotide(2'OMe-miR-21)isalsocalledmiRNAinhibitor.MiR-21inhibitor(2'OMe-miR-21)wassynthesizedbyIntegratedDNATechnologiesandhasthefollowingsequenceandstructure:5'mUmCmAmAmCmAmUmCmAmGmUmCmUmGmAmUmAmAmGmCmUmA-3'.Forthecontrolpurpose,wehaveusedtwocontrolsforthisstudy.Thefirstcontrolwasavehiclecontrol(PBS)andthesecondcontrolwasanirrelevant2'-O-methyl-oligonucleotide(2'OMe-EGFP).2'OMe-EGFPwasalsosynthesizedbyIntegratedDNATechnologiesandhasthefollowingsequenceandstructure:5'mAmAmGmGmCmAmAmGmCmUmGmAmCmCmCmUmGmAmAmGmU-3'..miR-21inhibitorTheantisLocaloligodeliveryintotheinjuredvascularwall.LocaloligodeliveryintotheTheeffectofmiR-21onneointimalformationafterangioplasty.TheeffectofmiR-21onneointWhatarethecellularmechanismsinvolvedinmiR-21mediatedeffectonneointimalformation?VSMCproliferationisthekeycellulareventforneointimallesionformation.ToidentifythecellularmechanisminvolvedinmiR-21-mediatedeffectonneointimallesionformation,wethereforedeterminedtheeffectofmiR-21inhibitor2'OMe-miR-21oncellproliferationinculturedVSMCsinthefollowingexperiments.ItiswellknownthatfreshlyisolatedVSMCsmimicdifferentiatedVSMCsinnormaluninjuredvascularwall,whereasserumculturedVSMCsmimicdedifferentiatedVSMCsinvascularneointimallesions.WethusdeterminedthemiR-21levelsinfreshlyisolated,differentiatedVSMCsanddedifferentiatedVSMCsculturedinDMEMcontaining10%FBS.WefoundthattheexpressionofmiR-21indedifferentiatedVSMCswassignificantlyhigherthanthatinfreshlyisolated,differentiatedVSMCs..Whatarethecellularmechanis2'OMe-miR-21decreasesthemiR-21expressionlevelinculturedVSMCsinadose-dependentmanner.2'OMe-miR-21decreasesthemiR2'OMe-miR-21decreasesVSMCproliferationinvitro.2'OMe-miR-21decreasesVSMCprRepresentativeBrdUstainedphotomicrographs(bottompanel)aswellastheircorrespondingtotalcellphotomicrographs(toppanel).RepresentativeBrdUstainedphWhatistheeffectofmiR-21onVSMCapoptosis?Neointimalgrowthisthebalancebetweencellapoptosisandcellproliferation.Thus,apoptosisisalsoanimportantcellulareventinneointimallesionformation.RecentreportsdemonstratedthatmiR-21hadanti-apoptosiseffectonglioblastomacells,buthadnoanti-apoptosiseffectonHeLacells.DoesmiR-21haveanti-apoptosiseffectonVSMCapoptosis?TodeterminetheeffectofmiR-21onVSMCapoptosis,weappliedawell-establishedVSMCapoptosismodelinwhichapoptosiswasmeasuredafter48hinserum-freeculture.ApoptosiswasevaluatedbydUTPnick-endlabeling(TUNEL)assayandcaspase-3activitymeasurement..WhatistheeffectofmiR-21oTheeffectofmiR-21onVSMCapoptosisinvitro.TheeffectofmiR-21onVSMCaRepresentativeTUNELstainedphotomicrographs(bottompanel)aswellastheircorrespondingtotalcellphotomicrographs(toppanel).RepresentativeTUNELstainedp2'OMe-miR-21decreasesVSMCproliferationinvivoinballooninjuredvessels.2'OMe-miR-21decreasesVSMCpr2'OMe-miR-21increasesVSMCapoptosisinvivoinballooninjuredvessels.2'OMe-miR-21increasesVSMCapPotentialgenetargetsinmiR-21-mediatedcellulareffectsonVSMCs

miRNAisabletobindtoitsmRNAtargetswitheitherperfectorimperfectcomplementarity.Thus,onemiRNAmayhavemultiplemRNAtargets.BasedonthecellulareffectsofmiR-21onVSMCs,weproposedthatmiR-21mighttarget,degradeatumorsuppressorgene,and/orblockitstranslation.

.PotentialgenetargetsinmiR-

computationalanalysisofmiR-21targets

ComputationalanalysissuggeststhatPTEN,Bcl-2,andtransforminggrowthfactor-beta(TGF-beta)mightbethepotentialtargetgenesformiR-21.ItisimportantthatallthecomputationalpredictedmiRNAputationalanalysisofmiRTGF-betaisnotagenetargetformiR-21.TGF-betaisnotagenetargetBcl-2isnotadirecttargetofmiR-21.Bcl-2isnotadirecttargetoPTENisadirecttargetofmiR-21

.PTENisadirecttargetofmiRSummaryoftheresults1.miRNAexpressionsignatureinarteryisidentifiedforthefirsttime.miRNAsareaberrantlyexpressedinthevascularwallswithneointimallesionformation.ModulatinganaberrantlyoverexpressedmiRNA,miR-21,viaantisense-mediateddepletion(knock-down)haveasignificantnegativeeffectonneointimallesionformation.

Invitro,theexpressionlevelofmiR-21indedifferentiatedvascularsmoothmusclecellswas