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1、·1534·2014年5月27日第94卷第2O期NatlMedJChina，May!．絲：．肝癌診治．小RNA靶向激活p21WAn基因表達(dá)對(duì)BEL一7402肝癌細(xì)胞增殖和凋亡的影響吳志明黃歡劉卓煒郭睜垮蔣麗娟董培賈國(guó)金陳剛【摘要】目的探討RNA激活技術(shù)上調(diào)p21基因的表達(dá)而影響人肝癌細(xì)胞株BEL-7402的增殖和凋亡能力。方法將與p21基因啟動(dòng)子區(qū)域DNA序列互補(bǔ)的RNA分子(dsP21—322)轉(zhuǎn)染入BEL一7402細(xì)胞中，采用qPCR法及Western印跡方法檢測(cè)p21“的表達(dá)；采用WST一1細(xì)胞增殖試劑檢測(cè)細(xì)胞增殖情況；使用流式細(xì)胞儀評(píng)估腫瘤細(xì)胞凋亡情
2、況并使用Western印跡方法檢測(cè)相關(guān)分子表達(dá)水平的變化。結(jié)果dsP21—322轉(zhuǎn)染72h后，BEL一7402細(xì)胞中p21”表達(dá)顯著上調(diào)；細(xì)胞增殖受到明顯抑制，凋亡率36．86％(細(xì)胞的體積變小、變形，細(xì)胞核固縮，染色質(zhì)凝聚呈深染，細(xì)胞出現(xiàn)皺縮、變圓、脫落)較對(duì)照組(11．51％、】4．06％)相比明顯升高。結(jié)論RNA激活技術(shù)靶向上調(diào)p21’基因表達(dá)并抑制細(xì)胞增殖、增加誘導(dǎo)腫瘤細(xì)胞凋亡，可作為肝細(xì)胞癌或其他惡性腫瘤基因治療的一種有效手段?！娟P(guān)鍵詞】癌，肝細(xì)胞；細(xì)胞增殖；p21“Activationofp21bysmallactivatingRNAinhibi
3、tscellproliferationandinducesapoptosisinBEL-7402hepatomacellWuZhiming，HuangHuan，LiuZhuowei，GuoZhengzheng，JiangL~uan，DongPei，JiaGuojin，ChenDepartmentofUrology，CancerCenter，SunYat—senUniversity，StateKeyLaboratoryofOncologyinSouthernChina，Guangzhou，Guangzhou510060，ChinaCorrespondingau
4、thor：ChenGn，DepartmentofUrology，JinshanHospital，F(xiàn)udanUniversity，Shanghai201508，China，Email：chgan305@163．com【Abstract】ObjectiveToevaluatetheantineoplasticeffectsofp21transcriptionalactivationinducedbyduplexRNAsinhepatocellulm’carcinonla(HCC)celllineBEL一7402．MethodsCe11sweretreatedwi
5、thdsRNAscomplementarytopronl0tersequeneesofp21w“．Quantitativepolymerasechainreaction(qPCR)andWesternblotwereemployedtodetecttheexpressionofp21．Atvarioustimepointspost—transfection，cellviabilityassayandapoptosisanalysiswereusedtodeterminetheeffectofRNAactivation．AtiertransfectionWes
6、ternblotwasa]soperformedtodetecttheexpressionofBcl—xL，cleavedcaspase一3．cleavedcaspase-9andcleavedPARP．ResultsDsP21—322transfectionsignificantlyinhibitedcellviability．And．a(chǎn)tDay5．dsP21—322inhibitedcellgrowthby65．84％versuscontro1．FlowcytometryrevealedthatdsP21—322causedasignificantinc
7、reaseofcellapoptosis．Thetotalpercentofapoptoticcells(UR+LR)increasedto36．86％versus11．51％and14．06％inmocksandcontrolSrespectively．Suchphenomenacorrelatedwithadecreaseofanti—apoptoticproteinBcl—xLandanincreaseofc】cavedcaspase一3．cleavedcaspase一9andcleavedPARP．ConclusionActivationofp21g
8、eneexpressionbysaRNAmayoff
