Survivin反义寡核苷酸对人卵巢癌细胞株中survivin和SMAC/DIABLO基因表达的影响

目的 观察survivin反义寡核苷酸(ASODN)对人卵巢癌细胞株SKOV3中survivin、Smac/DIABLO表达的影响,探讨survivin、Smac/DIABLO在ASODN诱导卵巢癌细胞凋亡和细胞周期改变中的作用和分子机制.方法 用脂质体(LipofectamineTM 2000)介导survivin ASODN转染人卵巢癌细胞株SKOV3,用MTT比色法检测细胞生长活性,流式细胞学技术检测细胞凋亡指数、细胞周期分布及survivin、Smac蛋白表达改变,逆转录酶链反应检测survivin、Smac/DIABLO基因表达改变.结果 同对照组比较,不同浓度ASODN作用后细胞生长明显减慢,转染后48 h IC50在600 nmol/1左右.用600 nmol/LASODN转染48h后,细胞凋亡指数(AI)明显增加,(t=6.3671,P＜0.05);更多的细胞停留在G0/G1期(t=10.3832,P＜0.01).Survivin mRNA和蛋白表达明显下调(t=3.5031,P＜0.05,t=7.8146,P＜0.01),SmacmRNA和蛋白表达上调(t=2.8011,P＜0.05,t=11.3917,P＜0.01).结论 ASODN诱导细胞凋亡,使更多的细胞停留在G0/G1期,减少进入有丝分裂细胞数的作用,是通过下调survivin基因,上调Smac基因表达来共同完成的,survivin、Smac/DIABLO基因在调节SKOV3细胞周期、凋亡的功能上密切相关,二者的相互作用是卵巢癌发生、发展的重要分子机制之一.

Effects of survivin antisense oligonucleotides on survivin and Smac gene expression in ovarian cancer cells

Objective To observe the effect of survivin antisense oligonucleotides(ASODN)on survivin and Smac gene expression in ovarian cancer SKOV3 cells and explore the role of survivin and Smac genes in ASODN-induced ovarian cancer cell cycle alteration and apoptosis and its molecular mechanisms.Methods Survivin ASODN was introduced via LipofectamineTM2000 into SKOV3 cells,whose growth activity was detected subsequently with MTT assay.The apoptosis index,cell cycle and changes in survivn and Smac protein expression were determined using flow cytometry.The changes in survivn and Smac mRNA expression were detected by RT-PCR.Results In comparison with the control cells,cells transfected with difference concentrations of survivin ASODN exhibited significantly inhibited growth,and 48 h after the transfection,the IC50 was about 600 nmol/L.After a 48-hour transfection of the cells with 600 nmol/L ASODN,the apoptosis index significantly decreased(t=6.3671,P<0.05),and the cell percentage in G1/G0 phase increased(t=10.3832,P<0.01),resulting also in significantly down-regulated survivin mRNA and protein expressions(t=3.5031,P<0.05;t=7.8146,P<0.01)and up-regulated Smac mRNA and protein expressions(t=2.8011,P<0.05;t=11.3917,P<0.01).Conclusion ASODN against survivin can induce apoptosis of ovarian cancer cell line SKOV3 and results cell growth arrest in G1/G0 phase by up-regulating Smac and down-regulating survivin expression.Survivin and Smac are closely correlated in their action on SKOV3 cell cycle and apoptosis,which is one of the important mechanisms of ovarian cancer development.