逆转录病毒载体介导的RNAi抑制Bmi-1基因表达对MCF-7细胞的影响
Effect of retrovirally mediated RNA in-terference targeting human Bmi-1 gene on MCF-7 cell line
武向梅 1卜友泉 1宋方洲 1刘涛 1张琴 1袁成福 1袁飞 1秦琴1
作者信息
- 1. 重庆医科大学重庆市分子医学与肿瘤研究中心生物化学与分子生物学教研室,重庆,400016
- 折叠
摘要
目的:研究BNA干扰(RNAi)抑制Bmi-1基因表达后对乳腺癌细胞株MCF-7凋亡和增殖的影响.方法:将表达Bmi-1短发夹RNA(shRNA)的重组逆转录病毒载体pSuper-retro/Bmi-1 si瞬时转染MCF-7乳腺癌细胞株,携带针对绿色荧光蛋白干扰序列的载体pSuper-retro/GFP si作为阴性对照.用RT-PCR和Western Blot分别从mBNA,蛋白水平检测Bmi-1的表达;MTT法检测细胞增殖;流式细胞术检测MCF-7细胞周期和凋亡的变化.结果:pSuper-retro/Bmi-1 si明显抑制MCF-7细胞Bmi-1 mRNA及蛋白表达.与空白对照组及阴性对照组相比较凋亡细胞数无明显增加;抑制Bmi-1基因表达使MCF-7细胞G1/S周期转换及细胞增殖速率受抑制.结论:表达shRNA的重组逆转录病毒载体pSuper-retro/Bmi-1 si能显著抑制乳腺癌细胞株MCF-7中Bmi-1基因表达,从而有效抑制MCF-7细胞增殖,为乳腺癌的基因治疗提供了实验依据.
Abstract
AIM: To investigate the inhibition effects of Bmi-1 (B-cell-specific moloney routine leukemia virus insertion site 1) with RNA interference (RNAi) on the apoptosis and proliferation of breast carcinoma MCF-7 cells. METHODS: Recombinant retroviral Bmi-1 gene short hairpin RNA(shRNA) expression vec-tor pSuper-retro/Bmi-1 si was transiently transfected into breast carcinoma MCF-7 cells and pSuper-retro/GFP si vector was used as negative control. Bmi-1 mRNA and protein of the transfected cells were examined with reverse transcriptianal polymerase chain reaction(RT-PCR) and Western blot assay, respectively. The proliferations of MCF-7 cells were examined by MTr and the cell cycle status and apoptosis of MCF-7 cells were detected by flow cytometry (FCM). RESULTS : pSuper-retro/Bmi-1 si retroviral vector resulted in effective inhibition of Bmi-1 mRNA and protein. Compared with that in uninfected control and negative control, no significant increase was observed in the basal levels of apoptosis in MCF-7 cells infected with pSuper-retro/Bmi-1 si. G1/S phase transformation and the growth of MCF-7 cells were significantly inhibited by Bmi-1. CONCLUSION: Retroviral shRNA expres-sion vector pSuper-retro/Bmi-1 si significantly inhibits the expres-sion of Bmi-1 and exerts effective inhibition effect on the viability of MCF-7 cells. The results of this study provide evidence for gene therapy for human breast cancers.
关键词
Bmi-1基因/重组逆转录病毒/RNA干扰Key words
Bmi-1 gene/recombinant retrovirus/RNA interfer-ence引用本文复制引用
出版年
2009
NETL
NSTL
维普
万方数据