【摘要】 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。更多还原

【Abstract】 Objective: To investigate the effect on proliferation,apoptosis and invasion of human ovarian carcinoma HO8910PM cells line by double-target RNA interference(RNAi) for Survivin and RhoA genes.Methods: The intervention vector containing Survivin-RhoA-microRNA was constructed and transfected into human ovarian carcinoma HO8910PM cells by liposome.HO8910PM cells transfected with blank vector was served as control group.After 48 hours,the expression of mRNA and protein of Survivin and RhoA were detected by RT-PCR and Western blot respectively;the growth of HO8910PM cells was analysis with MTT assay;The apoptosis rate was detected by flow cytometry;the invasion ability of HO8910PM cells were evaluated by Transwell assay.Results: The Survivin-RhoA-microRNA effectively down-regulated the expression of HO8910PM cells at both mRNA and protein levels.After 48 hours,the inhibition rate of Survivin mRNA and RhoA mRNA in experiment group were 68.82 % and 90.23 %,respectively;the inhibition rate of Survivin protein and RhoA protein in experiment group were 63.91 %,88.47 %,respectively.The MTT results showed that the A(OD490) of the experimental group was 0.706 ± 0.177,significantly decreased compared with those in the control group(1.172 ± 0.241)(P<0.05);Flow cytometry demonstrated that the apoptotic rate of cells in the experimental group was 36.41 ± 3.34 %,which increased compared with those in the rest groups(2.92 ± 0.78)(P<0.01).We also found that the cell invasion percentage was 12.56 ± 6.17 %,which significantly inhibited compared with those in the rest groups(32.96 ± 5.14 %)(P<0.05).Conclusion: The intervention vector containing Survivin-RhoA-microRNA was successfully constructed,which significantly inhibit the proliferation and invasion of human ovarian carcinoma HO8910PM cells,and it can also greatly promote their apoptosis,which has a synergistic effect and making a new way and strategy for ovarian carcinoma treatment.更多还原