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 interference targeting human Bmi-1 gene on MCF-7 cell line

目的:研究RNA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-1si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/GFPsi作为阴性对照.用RT-PCR和Western Blot分别从mRNA,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-1si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-1si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.

AIM： To investigate the inhibition effects of Bmi-1 （B-cell-specific moloney murine 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 vector pSuper-retro/Bmi-1 si was transiently transfccted 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 transcriptional polymerase chain reaction （ RT-PCR ） and Western blot assay, respectively. The proliferations of MCF-7 cells were examined by MTT 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 ceils 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 expression vector pSuper-retro/Bmi-1 si significantly inhibits the expression 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.