MiR-26aInhibitsCellGrowthandTumorigenesisof

NasopharyngealCarcinomathroughRepressionofEZH22011;71:225-233.CancerRes

是鱗狀上皮細(xì)胞癌，具有早期浸潤和遠(yuǎn)處轉(zhuǎn)移，惡性度高，其發(fā)生與遺傳易感性、環(huán)境因素及EB病毒感染相關(guān)，具有明顯的人種和地理分布的傾向性，在中國南方和東南亞高發(fā)，尤其是廣東地區(qū)最為多見。目前治療放療效果較好，5年生存率達(dá)到70%左右。鼻咽癌miRNAMicroRNA（miRNA）是近年來新發(fā)現(xiàn)的一類大小約為19~25個核苷酸（nucleotide,nt）的非編碼單鏈小分子RNA，由具有發(fā)夾結(jié)構(gòu)的大小約為70~100nt的單鏈RNA前體（pre-miRNA）經(jīng)過Dicer酶剪切加工后生成，通過與靶基因mRNA的3’非編碼區(qū)（3’untranslatedregion,3’UTR）互補(bǔ)匹配導(dǎo)致該mRNA的降解，大多數(shù)miRNA與底物mRNA不完全配對結(jié)合，通過轉(zhuǎn)錄后抑制翻譯來調(diào)控基因表達(dá)。miRNA基因以單拷貝、多拷貝或基因簇等多種形式存在于基因組中，而且絕大部分定位于基因間隔區(qū)，其轉(zhuǎn)錄獨(dú)立于其他基因，并不翻譯成蛋白質(zhì)，而是在體內(nèi)代謝過程中發(fā)揮多種調(diào)控作用，包括動植物的組織器官發(fā)育、細(xì)胞分化和凋亡、胰島素分泌、脂肪代謝等多種生命活動，其表達(dá)失調(diào)將導(dǎo)致重大疾病，如腫瘤的發(fā)生。研究發(fā)現(xiàn)miRNA在腫瘤的發(fā)病機(jī)制中發(fā)揮舉足輕重的作用。miRNA作為一類新型基因調(diào)控劑，與腫瘤的發(fā)生發(fā)展密切相關(guān)。有研究表明，50%以上的miRNA定位在腫瘤相關(guān)的基因組區(qū)域，包括LOH區(qū)、染色體擴(kuò)增區(qū)及脆性位點(diǎn)等，其表達(dá)水平在多種腫瘤中發(fā)生改變，通過調(diào)控下游靶基因的轉(zhuǎn)錄和翻譯，影響癌細(xì)胞的增殖、分化、凋亡、運(yùn)動、浸潤、轉(zhuǎn)移以及血管生成等生物學(xué)特性，發(fā)揮癌基因或抑癌基因的作用，多角度多層次的參與了腫瘤的演進(jìn)。miRNA在腫瘤中的作用miR-29c,miR-200a,miR-100,miR-141,miR-BART22,miR-BART5已證實與鼻咽癌的發(fā)生和進(jìn)展有關(guān)，miRNA芯片雜交提示miR-26a在鼻咽癌組織中表達(dá)下調(diào)，他人文獻(xiàn)miR-26a在膠質(zhì)瘤中為癌基因的作用，而在肝癌中則顯示為抑癌基因的作用。但miR-26a在鼻咽癌中的作用還沒有文獻(xiàn)報道。Figure1.TheexpressionofmiR-26awasreducedinNPCcelllinesandclinicalspecimens.A,expressionofmiR-26ain7NPCcelllines.B,averageexpressionlevelofmiR-26ainhumanNPCspecimens(n?18)andnormalnasopharyngealepithelialtissues(n?16).miRNAabundancewasnormalizedtoU6RNA.miR-26a在NPC細(xì)胞系和組織中的表達(dá)情況：表達(dá)下調(diào)(注：NP69為永生系細(xì)胞)Figure2miR-26a抑制NPC細(xì)胞生長和克隆形成C666-1HNE-1C666-1MTTFCMColonyformationassayFigure2.EnforcedexpressionofmiR-26ainducedgrowthinhibitioninNPCcells.A,effectofmiR-26aoncellproliferationwasmeasuredbyMTTassayaftermiRNAtransfectioninC666-1andHNE-1cells.B,representativehistogramsforcell-cycledistributionofC666-1cellstransfectedwithmiRNAsfor48

hours.C,expressionlevelsofmiR-26aafterC666-1andHNE-1cellswereinfectedwithLV-miR26aat5differentMOIs.D,representativepicturesofcolonyformationassayofLV-miR26a–infectedC666-1andHNE-1cells.E,colonieswereevaluatedandvalueswerereportedastheratiobetweenLV-miR26a-infectedcellsandLV-con–infectedcells.**,P<0.01comparedwithcontrol.Figure3

EZH2基因是miR-26a的靶基因HNE-1Figure3.EZH2wasadirecttargetofmiR-26ainNPCcells.A,expressionlevelsofEZH2afterLV-miR26ainfectionatdifferentMOIsinC666-1andHNE-1cells.B,expressionlevelsofmiR-26aandEZH2afterHNE-1cellsweretransfectedwithanti-miR26afor48hours.C,diagramofEZH230UTR–containingreporterconstructs.D,luciferasereporterassaysinC666-1cells,withcotransfectionofwtormt30UTRandmiRNAsasindicated.**,P<0.01comparedwithcontrol.After

LV-miR26aFigure4.EZH2wasinvolvedinmiR-26a-inducedgrowthinhibitioninNPCcells.A,C666-1cellswereinfectedwithLV-shEZH2orLV-miR26a.Cellgrowthrateandcell-cycledistributionweremeasuredbyMTTassayandflowcytometry.B,C666-1cellswereinfectedwithLV-miR26afor72hours,followedbyinfectionwithLV-EZH2,andMTTassayandcell-cycleanalysiswerethenperformed.**,P<0.01comparedwithcontrolorcomparisonbetween2groupsasindicated.C666-1EZH2過表達(dá)可以拮抗miR-26a的抑制作用細(xì)胞周期相關(guān)基因：EZH2上調(diào)的基因與miR-26a下調(diào)的基因具有一致性，EZH2能拮抗miR-26a的作用

Figure5.Cell-cycleregulatorscontributedtothegrowthinhibitioninducedbymiR-26a.A,deregulatedexpressionof14cell-cycleregulatorsinC666-1cellsaftermiR-26amimicorinhibitortransfection.B,theirdysregulatedexpressioninC666-1cellsafterLV-EZH2orLV-shEZH2infection.C,EZH2overexpressionrescuedthederegulatedexpressionofmiR-26aeffectorsinC666-1cells.D,adiagramofmiR-26asignalingpathwaysforcell-cyclecontrolinNPCcells.miR-26a過表達(dá)和EZH2沉默均可抑制CCND3、CCNE2、CDK4、CDK6和c-myc，同時上調(diào)p14ARF和p21CIP1，而對CCND1、CCNE1、CDK2、p16INK4A、p53和pRb的表達(dá)無影響。Figure6.MiR-26asuppressedtumorgrowthofNPCcellsinnudemice.A,C666-1cellswereinfectedwithLV-miR26aandinjectedsubcutaneouslyintonudemice.At2weeksafterimplantation,LV-miR26a-infectedcellsproducedsmallertumorsthancontrolcells.B,representativepictureoftumorsformed.C,growthcurveoftumorvolumes.EachdatapointrepresentsthemeanSEMof5mice.D,Ki-67-andPCNA-stainedsectionsoftransplantedtumorsformedbyC666-1cellsinfectedwithLV-miR26aat2weeksafterorthotopictransplantation.Magnification,200
.*,P<0.05;**,P<0.01comparedwithcontrol.miR-26a可以抑制NPC的成瘤能力C666-1Figure7.EZH2wasupregulatedinNPCspecimensandinverselycorrelatedwithmiR-26alevels.A,

averageexpressionlevelofEZH2inhumanNPCspecimensandnormalnasopharyngealtissues.EZH2abundancewasnormalizedtoGAPDH.B,astatisticallysignificantinversecorrelationbetweenmiR-26aandEZH2mRNAlevelsinNPCspecimens(Spearman'scorrelationanalysis,r?0.831;P<0.001).EZH2在NPC組織中表達(dá)升高EZH2與miR-26a表達(dá)的負(fù)相關(guān)小結(jié)miR-26a在鼻咽癌組織和細(xì)胞系中表達(dá)下調(diào)，上調(diào)miR-26a表達(dá)可以通過G1細(xì)胞周期阻滯顯著抑制細(xì)胞增生和克隆形成。在鼻咽癌細(xì)胞中，miR-26a明顯降低癌基因EZH2基因表達(dá)，EZH2表達(dá)下調(diào)抑制細(xì)胞生長和細(xì)胞周期進(jìn)展，EZH2的過表達(dá)可以對抗miR-26a的抑制效應(yīng)。機(jī)制研究表明m