Gli1表达抑制对卵巢癌SKOV3细胞增殖的影响

目的 抑制Glioma-associated oncogene homoglog（GLI）基因在卵巢癌SKOV3细胞中的表达,探讨GLI1表达抑制对SKOV3细胞增殖的影响.方法 构建并筛选GLI1基因的RNAi表达质粒,转染SKOV3细胞,CCK-8法比较转染前后卵巢癌细胞增殖变化,流式细胞仪检测紫杉醇诱导转染前后细胞凋亡比例,流式细胞仪检测转染前后SKOV3的细胞周期情况.Westem blot检测cyclinD、Bcl-2及Capase3蛋白表达的影响.结果 转染48 h后,在SKOV3细胞中Control组、N-Control组、GLI1 shRNA-1组、GLI1 shRNA-2组、GLI1shRNA-3组、GLI1 shRNA-4组的GLI1 mRNA水平分别为1.00±0.00、1.03 ±0.02、0.73±0.07、0.98 ±0.08、0.53±0.08、0.68±0.04,与Control组相比,GLI1 shRNA-1、GLI1 shRNA-3、GLI1 shRNA-4组GLI1 mRNA表达量均下降（P＜0.05）,转染GLI1 shRNA-3的SKOV3细胞中GLI1蛋白表达水平低于GLI1 shRNA-1、GLI1shRNA-2、GLI1 shRNA-4组（P＜0.05）,具有显著的干扰效果.转染GLI1 shRNA-3的SKOV3细胞增殖受到明显抑制（P＜0.05）,在紫杉醇的诱导下细胞凋亡比例明显增加,细胞周期主要阻滞在G1/S期.Western blot检测发现Bcl-2蛋白表达下降（P＜0.05）,Capase3蛋白表达升高（P＜0.05）,细胞周期蛋白CyclinD1蛋白表达下降（P＜0.05）.结论 GLI1 shRNh对SKOV3细胞增殖抑制作用明显,促进细胞凋亡,GLI1信号可通过抑制cyclinD1活化抑制细胞恶性增殖.

Construction of GLI1 shRNA expression vector and effects on the proliferation of ovarian cancer cell

Objective To explore the effects of GLII signaling on the proliferation of ovarian cancer cell.Methods Ovarian cancer SKOV3 cells were transfected for 48 hours by the mediation of LipofectamineTM 2000.CCK-8 kit and real time RT-PCR were employed to study the effects of GLI1 mRNA transfection on the proliferation of SKOV3 cell,the apoptosis rate induced by paclitaxel detected by flow cytometry,the cell cycle of SKOV3 cells following the treatments was measured with flow cytometry and the expression of cyclinD1 protein detected by western blot.Results In the Control,negative-control group,GLI1 shRNA-1 group,GLI1 shRNA-2 group,GLI1 shRNA-3 group,GLI1 shRNA-4 group,of SKOV3 cells,the expression of GLI1 mRNA relative to GAPDH was 1.00 ±0.00,1.03 ±0.02,0.73 ±0.07,0.98 ±0.08,0.53 ±0.08,0.68 t0.04,respectively.Contrast to Control group,the GLI1 mRNA expression in GLI1 shRNA-1 group,GLI1 shRNA-3 group,GLI1 shRNA-4 group declined （P 〈 0.05）,moreover the GLI1 mRNA expression in GLI1 shRNA-3 group obviously lower than other 3 groups （P 〈 0.05）.Meanwhile the expression of GLI1 protein decreased.SKOV3 cell proliferation and CyclinD1 protein significantly inhibited under the condition of transfected by GLI1 shRNA-3.The apoptosis rate increased induced by paclitaxel after GLI1 down-regulated.The expression of Bcl-2 and CyclinD1 were decreased and the expression of Caspase3 was increased.Conclusions GLI1 shRNA visibly inhibit the proliferation of ovarian cancer cell and induce the cell apoptosis,GLI1 signaling could induce cell proliferation by actived CyclinD1.