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1、肿瘤2009年4月第29卷第4期TumorVol.29,No.4,April,2009·345·DOI:10.3781/j.issn.100027431.2009.04.011BasicResearch·基础研究livin基因沉默对人恶性黑素瘤A375细胞周期和凋亡的影响王琳琳,郑洪,唐薇薇,厉国慧,安文波(遵义医学院附属医院病理科,遵义563003)[摘要]目的:探讨小干扰RNA(smallinterfeningRNA,siRNA)沉默livin基因对人恶性黑素瘤A375细胞周期和凋亡的影响。方法:构建2个针对人源livin基因的siRNA表达质粒,并通过脂质体介导
2、转染人恶性黑素瘤A375细胞株,利用荧光定量PCR检测livinmRNA的表达情况,AnnexinⅤ2PEFCM法检测A375细胞凋亡情况,PI单染FCM法检测细胞周期情况。结果:成功构建livinsiRNA表达质粒,转染siRNA21和siRNA22后48h的A375细胞中livinmRNA表达量较空白对照组分别下降17%和42%,72h分别下降71%和61%。转染siRNA21和siRNA22后48h的A375细胞凋亡率(29.45%和33.25%)及72h细胞凋亡率(33.52%和38.18%)均高于空白组(7.95%和9.70%)(P<0.05)。siRNA2
3、2可引起G0/G1期升高,S期下降(P<0.05),siRNA21作用不明显(P>0.05)。结论:siRNA21、2体外均能有效抑制livinmRNA的转录,促进人恶性黑素瘤A375细胞凋亡。siRNA22表达质粒能使人恶性黑素瘤A375细胞周期出现G0/G1期阻滞,细胞增殖受到抑制。[关键词]黑色素瘤,实验性;RNA干扰;细胞凋亡;细胞周期;基因,livin[中图分类号]R739.5[文献标志码]A[文章编号]100027431(2009)0420345205EffectsoflivingenesilencingbysiRNAonapoptosisandcellc
4、ycleofhumanmalignantmelanomaA375cellsWANGLin2lin,ZHENGHong,TANGWei2wei,LIGuo2hui,ANWen2bo(DepartmentofPathology,AffiliatedHospitalofZunyiMedicalCollege,Zunyi563003,China)[ABSTRACT]Objective:ToexploretheeffectsoflivingenesilencingbysmallinterfeningRNA(siRNA)ontheapoptosisandcellcycleofhu
5、manmalignantmelanomaA375cells.Methods:TwosiRNAeukaryoticexpressionvectortargetingthelivingenewerecon2structedbygenerecombination,andthentransfectedintohumanmalignantmelanomaA375cellsmediatedbyliposome.ExpressionleveloflivinmRNAwasassayedbyreal2timefluorescencequantitativePCRtechnology.T
6、heapoptosiswasdetectedbyAnnexinV2PEflowcytometry(FCM)andthecellcyclewasdeterminedbyFCMwithsinglePIstaining.Results:Theexpressionvectorstargetinglivinweresuccessfullyconstructed.ExpressionoflivinmRNAinA375cellswasdecreasedby17%and42%after482htransfectionofsiRNA21andsiRNA22comparedwiththe
7、blankcontrolgroup;anddecreasedby71%and61%after722htransfection.TheapoptoticratiosofA375cellswere29.45%and33.25%after482htransfectionofsiRNA21andsiRNA22,respectively.Theywere33.52%and33.18%after722htransfectionofsiRNA21andsiRNA22,respectively.Allwerehigherthanthoseinblankcontrol
