RNAi抑制人大肠癌LOVO细胞Bmi-1基因的表达

目的探讨RNA干扰（RNAi）技术沉默Bmi-1基因表达对人大肠癌细胞株LOVO增殖和侵袭力的影响及机制。方法将化学法合成的针对Bmi-1mRNA不同位点设计的3对siRNA序列（siR-NA1～3）和1对带有荧光标记的FAM-siRNA（siRNA4）转染至人大肠癌LOVO细胞,荧光显微镜下观察siRNA的转染效率,QRT-PCR检测Bmi-1mRNA表达抑制作用,Western Blot检测Bmi-1蛋白表达变化,MTT法检测LOVO细胞体外增殖变化,小室侵袭实验检测LOVO细胞侵袭能力。结果利用荧光标记的Oligo观察到siRNA转染效率达70%。siRNA1对mRNA表达抑制率最高,从而筛选出沉默效应最强的siRNA序列为siRNA1。siRNA1转染组LOVO细胞Bmi-1蛋白表达,增殖及侵袭力明显低于阴性对照组和空白对照组（P〈0.05）。结论化学合成的靶向Bmi-1siRNA转染人大肠癌LOVO细胞能有效抑制Bmi-1的mRNA和蛋白质表达水平,Bmi-1基因的RNA干扰可有效抑制人大肠癌LOVO细胞的增殖和侵袭能力。

Effects of RNAi on expression of Bmi-1 gene in human colorectal carcinoma LOVO cells

Objective To investigate the mechanism and effect of small interfering RNA (siRNA) on Bmi-1 in the human colorectal carcinoma LOVO cells. Methods Three siRNA sequences targeting Bmi-1 (siRNA1, siRNA2, siRNA3), a fluorescein-labeled double-stranded RNA (dsRNA) oligomer (siRNA4), and the negative control were chemically synthesized. All siRNA sequences were transinfected into LOVO cells. Inverse microscopy was used to observe the efficacy of transinfecion. Bmi-1 mRNA expression and protein expression were detected by real time quantitative PCR (QRT-PCR) and Western blot, respectively. The proliferation and invasion of LOVO cells were detected by MTT and transwell invasion assay, respectively. Results The efficacy of transfection was 70%. The Bmi-1 mRNA expression inhibition rate of siRNA1 was the most effective, and therefore was selected to interfere with the expression of Bmi-1 gene. The Bmi-1 protein expression and cell proliferation and invasion were significantly fewer than that in the control group and the blank group, P < 0.05. Conclusions The siRNA targeting on Bmi-1 gene could not only inhibit the Bmi-1 mRNA and protein expression in the human colorectal carcinoma LOVO cells, but also could restrain the proliferation and invasion of human colorectal carcinoma LOVO cells.