miR34.docx
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miR34.docx
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miR34
miR-34cenhancesmousespermatogonialstemcellsdifferentiationbytargetingNanos2+
Abstract
MiRNAsareexpressedinmanymammaliancells,actingspecificrolesinregulatinggeneexpressionormediatingspecialmRNAscleavagebytargetingtheir3'UTR.SomemiRNAsareessentialandimportantforanimaldevelopment.However,itisstillunclearwhattherelationshipisbetweenmiR-34candmammalianspermatogonialstemcells(SSCs).WefoundthataconservedmicroRNA-34cthroughitstarget-Nanos2,regulatingSSCs’differentiationinmouse.ImmunohistochemistryanalysisofNanos2andmiR-34cFISHresultsrevealedtheoppositeexpressiontrendsbetweenthem.SevenbioinformaticswebsitesandprogramspredictedthatmiR-34chasinteractionsitesinNanos2’s3'untranslatedregion(3'UTR).Dual-luciferasereportervectorandmutateddual-luciferasereportervectoranalysisvalidatedthattheyareinteracted.AftertransfectionmiR-34cmimicsintomouseSSCs,ormiR-34clentiviralvectorinvitroco-cultivationwithseminiferoustubules,andwesternblotanalysisdemonstratedthatmiR-34cover-expressioncouldsuppressNanos2expressioninpost-transcriptionlevel.OurexperimentsidentifiedthatmiR-34cmaypromotemeiosisprocessbyinteractingwithNanos2leadingup-regulationofStra8inmousespermatogonialstemcells.
MiRNA在很多哺乳动物细胞中表达,在调节基因表达或靶向作用mRNA的3'非编码区域(UTR)介导特定mRNA分裂起着特殊作用。
一些MiRNA对动物的发育是必须而又非常重要的。
然而仍然不清楚miR-34和哺乳动物精原干细胞(SSCs)之间的关系。
在老鼠中,我们研究发现miR-34c靶向作用Nanos2+调节精原干细胞的分化。
免疫组化分析Nanos2和miR-34c的FISH结果揭示了它们之间的表达趋势相反。
七个生物信息学网址和程序预测miR-34c与Nanos2+的3'UTR相互作用。
双荧光素酶报告载体和突变双荧光素酶报告载体分析证实了它们能够相互作用。
在miR-34c类似物转染进SSCs或miR-34c慢病毒载体与细精管在体外共培养,westernblot分析证明miR-34c过表达可以抑制Nanos2在转录后水平的表达。
我们的实验证实,在老鼠精原干细胞中,miR-34c通过与Nanos2+相互作用可能促进减数分裂导致Stra8的上调。
Introduction
Spermatogenesisisahighlymediatedprocessofgermcelldifferentiation.Takingprotamine(Prm1)asanexample,meiosisisknownfortheirhightranscriptionalandlowtranslationalactivitiesduringspermatogenesisinmalegermlinestemcells(mGSCs).Therefore,post-transcriptionalregulationissignificantformammalianspermatogonialstemcells(SSCs)[Wuetal.,2011].MicroRNAs(miRNAs)areinvolvedinnearlyeverybiologicalprocessexaminedtodate,butlittleisknownoftheidentityorfunctionofmiRNAsintheirpotentialinvolvementinspermatogenesis[Curryetal.,2011;Huaetal.,2010].TherearesomemiRNAs,whichplaycriticalrolesintheprocessofspermatogenesis.Forinstance,miR-383isassociatedwithmaleinfertilityandpromotedembryonaltesticularcarcinomacellproliferation[Lizeetal.,2010].MicroRNA-184down-regulatesnuclearreceptorco-repressor2inmousespermatogenesis[Wuetal.,2011].Additionally,miR-34familyisconservedamongvariousspecies,functioningintheprocessesofproliferation,apoptosis,anddifferentiation[Corneyetal.,2007].MiR-34ccouldplayanessentialroleinlatespermatogenesisprocess[Bouhallieretal.,2010].In2011,BrinsterandhiscolleaguesfoundthatmiR-34cprohibitedthemostabundantlyinSSC-enrichedgermcellculturesbysmallRNAlibrariesconstructionandanalysis[Niuetal.,2011].ThesestudieshighlightedtheimportanceofmiR-34cexpressionincontrollingSSCs’growthanddifferentiation.SSCsarenecessaryforspermatogenesis,althoughtheyconstituteoneinthousandcellsintestis[Niuetal.,2011].NANOS2belongstoNanosfamilywhichcontainsevolutionarilyconservedzinc-fingermotifencodingRNA-bindingproteins,requiredinmouseSSCsformaintainingtheirself-renewalbypreventingdifferentiation[Shenetal.,2010].Inmousemalegermlinestemcells(mGSCs),Nanos2suppressesmeiosisandinturnisrequiredinmaintainingSSCs[Suzukietal.,2008;Sadaetal.,2012].Stra8isaprovedsignatureofenteringmeiosisinbothmaleandfemalegermcells;Nanos2cansuppresstheexpressionofStra8inmousestemcells[Suzukietal.,2008;Cannelletal.,2010].miR-34ccouldup-regulateStra8expressionindairygoatmGSCs[Lietal.,2013].Nanos2interacts4withCnot1,acomponentofCCR4-NOTdeadenylationcomplex,(TheCcr4-Notcomplexisaunique,essentialandconservedmulti-subunitcomplexthatactsatthelevelofmanydifferentcellularfunctionstoregulategeneexpression)whichcouldbeco-localizedwithPbodies.NANOS2-interactingRNAsmayberecruitedtoP-bodiesanddegradedbytheenzymescontainedthereinthroughNANOS2-mediateddeadenylation[Cannelletal.,2010;Liangetal.,2012].
InPbodies(processingbodies),Dicerenzymesrecognizespecificdouble-strandedRNA,producingsmallfragmentRNAwhose3’endhastwoprominentbases.Double-strandedmiRNAsnucleasecombinetoformtheRNA-inducedsilencingcomplex(RISC),miRNAsopendouble-strandedtoactivateRISCbybasepairingwithmRNAcombination,thenmakemRNAdecayortranslationalrepression[Olszewskaetal.,2012].However,therewerelittleinformationonmiR-34effectonmouseSSCsandtherealmechanism.ToexploretherelationshipbetweenNanos2,miR-34candStra8,weinvestigatedtheexpressionpatternsofmiR-34candfoundthatmiR-34ccouldplaycriticalrolesinregulationofmSSCs’meiosisdifferentiationthroughsuppressionitstarget-Nanos2,simultaneously,up-regulationofStra8,Scp3.
Results
CharacterizationofSSCsderivedfrom6-12dpostnatalmousetestis
SSCswerederivedfrom6to12dpostnatalKunmingmouse.Atfirst,theculturedSSCswerepresentedpairedoraligned,or4to8singlecellsaggregatedcolonies(Fig.1A).PCRindicatedthattheculturedSSCswerepositiveforOct4,CD90,Nanos2,whileMEFcellswerenegativefortheSSC’smarkers(Fig.1B).Theyformedtypicalcoloniesat2ndpassage.From3rdor4thpassages,SSCswereplatedontoMEFlayers.MostSSCswerepositiveforGFRα1,CD90,NANOS2,PLZF(spermatogonialstemcellmarkers)byimmunofluorescence(IF)assay.VASApositivestainingdemonstratedthatSSCspresentedmalegermcellscharacters(Fig.1C).
MiR-34cwashighlyexpressedintheadultmousetestis
TolocalizemiR-34cexpressioninthedevelopingtestis,amiR-FISHprobein14dpp,21dpp,and28dppandadultmousetestiswereused.Ascrambledprobewasusedasanegativecontrol.TheresultsshowedthatthemiR-34csignalexhibitedstrongest,andthepercentageofmiR-34cpositivespermatogoniareachedthetopinadultmurinetestis(Fig.2A,B).Incontrast,thesignalintensityandthepercentageofmiR-34cpositivein2dpp,7dppmousetestisweresignificantlyweakercomparedthanthatinadulttestis(datanotshown).AccordingtotheFISHresults,intheadultmousetestis,thehybridizationsignalformiR-34cwasdetectedinpachytenespermatocytesandroundspermatids(arrowheadsinFig.2A).Theseresultswereinconsistentwithpreviousstudies[Bouhallieretal.,2010;Liangetal.,2012;Zhangetal.,2012].
NANOS2isadirecttargetofmiR-34c
InordertoexplorehowmiR-34cregulatesmSSCs,wecomputationallypredictedthatNanos2wasthecandidateofmiR-34ctargetsfrommiRwalkdatabase(http:
//www.ma.uni-heidelberg.de/apps/zmf/mirwalk/).ThenmiRDB(http:
//mirdb.org/miRDB/)providedthedetailedinteractioninformationbetweenmiR-34candNanos2(Fig.3A,11B).Itwasvalidatedthattheydidhaveinteractionanalyzedbyluciferasereporterassay.Thepredictedbindingsite,3'UTRofNanos2wastheninserteddownstreamfromtheRenillaluciferasecodingregioninthereportervector(Fig.3C).Eachreporterconstructwasseparatelyco-transfectedwiththemiR-34cmimicsintoHelacells.Comparedtothemut-Nanos2-3'UTRcontrol,theluciferaseactivitydeclinedbyabout37.5%aftertransfectionwithmiR-34cmimicsandNanos2-3'UTRreportervector(Fig.3D).Theluciferaseanalysisshowedthatectopicover-expressionofmiR-34creducedNanos2proteinexpressionviadirectlybindingtoNanos23'UTR,indicatingthatNanos2isonetargetofmiR-34c.TheseresultsdemonstratedthatmiR-34cdirectlyregulatesNanos2proteinexpressionthroughitsbindingtothe3'UTRregionofNanos2.
MiR-34cover-expressioninhibitedNanos2,andpromotedmeiosisinmSSCs
TofurtherinvestigatetheeffectsofmiR-34conmSSCs,negativecontrolsmallRNAs,miR-34cmimic,miR-34cinhibitor,andincombinationwithmiR-34cmimicanditsinhibitorweretransfectedintomSSCs,QRT-PCRresultsmanifestedthatmiR-34cweretransfectedefficientlyintoSSCs(Fig.4A),andover-expressionmiR-34cspecificallydown-regulateditstarget-Nanos2.Simultaneously,themRNAexpressionlevelsofNanos3,Stra8(thepre-meioticmarkers)andScp3(meioticmarker)wereup-regulatedbymiR-34cover-expressionat48haftertransfectionintomSSCs(Fig.4B).Furthermore,wefoundthatmSSCstransfectedmiR-34cmimicsbecomeirregularedgedcomparedwiththattransfectedNC.ImmunofluorescenceanalysisrevealedtheexpressionlevelofmiR-34c’starget-NANOS2wassignificantlydown-regulatedbymiR-34c,andthepre-meioticmarkerSTRA8andmeiotic,germcellmarkerSCP3weresignificantlyup-regulatedinover-expressionofmiR-34ccomparedwithNC.Additionally,germcellmarker-VASAwaslittleup-regulated,however,PLZF(self-renewalmarkerofSSCs)wasdown-regulatedbymiR-34c(Fig.5A,B).Nanos2siRNAanalysisshowedthatthemeiosismarkers:
Stra8andScp3expressionlevelswerespecificallyupregulatedinNanos2siRNAgroupcomparedwithcontrolanalysedbyQRT-PCRandimmunofluorescence(SupplementedFig.1,2).12
MiR-34cover-expressioneffectinseminiferoustubules
ToassesshowmiR-34cfunctioninvivo,lentiviralparticlesofpLL3.7-CMV-34cwereculturedwithmouseseminiferoustubules[Chuetal.,2012].Throughimmunofluorescencemicroscope,thetransducedGFPpositivecellswereobservedinseminiferoustubules(Fig.6A).PCRanalysisandwesternblotshowedthattheexpressionofScp3andStra8inmRNAandprote
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