RNAi沉默Survivin与ER-α36基因对乳腺癌MDA-MB-231细胞增殖与凋亡的影响

Effect of silencing Survivin and ER-α36 genes with RNA interference on proliferation and apoptosis of human breast cancer cell MDA-MB-231

目的:靶向沉默人乳腺癌细胞Survivin与雌激素受体-α(ER-α)36双基因的表达,探讨其对乳腺癌MDA-MB-231细胞增殖与凋亡的影响。方法:采用生物信息学技术设计并构建RNAi片段(siRNA-ER-α36),转染乳腺癌MDA-MB-231细胞株。采用噻唑蓝染色法检测细胞增殖情况。采用RT-PCR、Western blot方法检测细胞中Survivin、ER-α36与凋亡蛋白Caspase-3的mRNA及蛋白表达情况;流式细胞术检测转染细胞凋亡率。结果:RNAi片段成功转染MDA-MB-231细胞,24 h转染率的平均值为68%,48 h转染效率可达76%(P<0.01)。与空白对照组比较,RNAi片段转染后MDA-MB-231细胞Survivin和ER-α36基因的mRNA和蛋白含量明显下降(P<0.05),而上调Caspase-3凋亡蛋白的表达;MDA-MB-231细胞增殖能力降低(P<0.05),凋亡率升高(P<0.05)。结论:RNAi靶向沉默并下调Survivin与ER-α36基因的表达,上调Caspase-3凋亡蛋白的表达,从而抑制乳腺癌MDA-MB-231细胞增殖,促进细胞凋亡,提示RNAi沉默可成为对乳腺癌相关基因功能研究的主要技术之一。

Objective:To explore the effect of RNA interference on human breast cancer MDA-MB-231 cell proliferation and apoptosis by inhibiting Survivin and ER-α36 double expression.Methods:RNAi fragment(siRNA-ER-α36)was designed and constructed by bioinformatics technology and transfected into breast cancer MDA-MB-231 cell line.Cell proliferation was detected by thiazol blue staining.RT-PCR and Western blot were used to detect the mRNA and protein expression of Survivin,ER-α36 and apoptosis protein Caspase-3.The apoptosis rate of transfected cells was detected by flow cytometry.Results:RNAi fragments were successfully transfected into MDA-MB-231 cells,the average transfection rate of 24 h was 68%,and the transfection efficiency of 48 h was 76%(P<0.01).Compared with the blank control group,the mRNA and protein contents of Survivin and ER-α36 genes in MDA-MB-231 cells transfected with RNAi fragment were significantly decreased(P<0.05),while the expression of Caspase-3 apoptosis protein was up-regulated.The proliferation ability of MDA-MB-231 cells was decreased(P<0.05),and the apoptosis rate was increased(P<0.05).Conclusion:RNAi targeted silencs and down-regulats the expression of Survivin and ER-α36 genes,up-regulats the expression of Caspase-3 apoptosis protein,thereby inhibits the proliferation of breast cancer MDA-MB-231 cells and promots cell apoptosis,suggesting that RNAi silencing can become one of the main technologies for the study of breast cancer related gene function.