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目的:探讨RNA干扰(RNAi)双向沉默Survivin和RhoA基因表达对人卵巢癌HO8910PM细胞增殖、凋亡及侵袭能力的影响。方法:构建Survivin和RhoA基因的串联microRNA干扰载体(Survivin-RhoA-microRNA),通过脂质体介导转染人卵巢癌HO8910PM细胞作为实验组,对照组为空载体转染组。转染48小时后,RT-PCR检测Survivin和RhoA的mRNA表达,Western-Blot检测Survivin和RhoA的蛋白质表达;利用MTT法、Transwell小室体外侵袭实验及流式细胞术检测转染后卵巢癌细胞的增殖、侵袭及凋亡情况。结果:Survivin-RhoA-microRNA明显抑制了HO8910PM细胞中Survivin和RhoA基因mRNA和蛋白的表达:转染48小时后,实验组Survivin mRNA和RhoA mRNA表达抑制率分别为68.82%、90.23%,Survivin和RhoA蛋白表达抑制率分别为63.91%、88.47%;MTT分析显示实验组细胞OD490的值为0.706±0.177,较对照组(1.172±0.241)明显降低,差异有统计学意义(P<0.05);流式细胞术检测实验组细胞凋亡率为36.41±3.34%,较对照组(2.92±0.78%)明显升高(P<0.01);实验组细胞侵袭百分比为12.56±6.17%,较对照组(32.96±5.14%)明显降低(P<0.05)。结论:成功构建Survivin和RhoA串联microRNA干扰载体(Survivin-RhoA-microRNA)。Survivin和RhoA双基因沉默显著抑制了卵巢癌HO8910PM细胞的增殖和侵袭能力,并增加其凋亡率,两者具有协同作用,为双靶点治疗卵巢癌提供了新的思路和策略。
OBJECTIVE: To investigate the effects of RNA interference (RNAi) bidirectional silencing Survivin and RhoA gene on the proliferation, apoptosis and invasion of human ovarian cancer HO8910PM cells. Methods: Survivin and RhoA gene were cloned into the vector of survivin-RhoA-microRNA and transfected into human ovarian cancer cells HO8910PM by lipofectamine. The control group was transfected with empty vector. Forty-eight hours after transfection, the mRNA expression of Survivin and RhoA was detected by RT-PCR, and the protein expression of Survivin and RhoA was detected by Western-Blot. MTT assay, Transwell chamber invasion assay and flow cytometry were used to detect the expression of Survivin and RhoA Proliferation, Invasion and Apoptosis. RESULTS: After Survivin-RhoA-microRNA significantly inhibited the expression of Survivin and RhoA mRNA and protein in HO8910PM cells, the inhibitory rates of Survivin mRNA and RhoA mRNA in experimental group were 68.82%, 90.23%, Survivin and RhoA The inhibitory rates of protein expression were 63.91% and 88.47% respectively. MTT assay showed that the OD490 value of the experimental group was 0.706 ± 0.177, which was significantly lower than that of the control group (1.172 ± 0.241) (P <0.05) The cell apoptosis rate in experimental group was 36.41 ± 3.34%, which was significantly higher than that in control group (2.92 ± 0.78%) (P <0.01). The percentage of cell invasion in experimental group was 12.56 ± 6.17%, which was 32.96 ± 5.14%) was significantly lower (P <0.05). Conclusion: Survivin and RhoA tandem microRNA interference vector (Survivin-RhoA-microRNA) was successfully constructed. Survivin and RhoA double gene silencing significantly inhibited the proliferation and invasion of ovarian cancer HO8910PM cells and increased the apoptosis rate. Both of them had synergistic effects and provided new ideas and strategies for the treatment of ovarian cancer.