应用RNAi敲减MARCH6基因对乳腺癌MCF-7细胞增殖及周期的影响

Inhibition of cellular proliferation by knockdown of MARCH6 gene expression in breast cancer cells

目的:应用慢病毒介导的RNAi技术,在乳腺癌MCF-7细胞中敲减MARCH6基因的表达,研究其对MCF-7细胞增殖及细胞周期的影响。方法:靶向MARCH6基因的sh RNA慢病毒转染293T细胞后,在荧光显微镜下观察转染效率;收集慢病毒液感染MCF-7细胞后,应用实时荧光PCR和Western印迹验证RNAi敲减MARCH6基因后,MARCH6基因m RNA和蛋白的表达影响;应用MTT,Brd U细胞增殖实验检测细胞增殖能力的变化;采用增殖指数染色流式细胞术检测细胞周期和增殖的改变。结果:通过在荧光显微镜下观察,MARCH6 sh RNA慢病毒质粒成功转染293T细胞,与同明视野对比,可见80%左右的细胞带绿色荧光;感染MCF-7细胞,90%的细胞均带绿色荧光;实时荧光PCR和Western印迹验证了MARCH6 sh RNA慢病毒感染MCF-7细胞后,MARCH6在转录水平和蛋白水平表达均显著下降;MTT实验、Brd U法结果显示MARCH6基因敲减组的MCF-7细胞增殖显著下降(P<0.01),流式细胞术分析显示MARCH6敲减后MCF-7细胞出现细胞周期抑制,细胞分裂处于G1期细胞数明显增多,增殖指数显著降低。结论:敲减乳腺癌MCF-7细胞的MARCH6基因表达后,抑制了MCF-7细胞的增殖,并引起细胞周期G1期阻滞。提示MARCH6通过影响细胞周期的进展,促进乳腺癌细胞的增殖。

Objective： To investigate effects of MARCH6 gene knockdown on MCF-7 cell proliferation and cell cycle. Methods： 293T cells were transfected with MARCH6 shRNA lentivirus. Fluorescence microscope was used to observe and verify the transfection efficiency. The initial effect of the MARCH6 gene knockdown in MCF-7 cells was observed via fluorescence microscope. Real-time PCR and Western blot were used to detect the expression of MARCH6. MTT and BrdU assay were used to examine cell proliferation, and staining flow cytometry was used to analyze cycle distribution of MCF-7 cells.Results： MARCH6 shRNA lentivirus was successfully transfected and about 80% of the cells expressed green fluorescent in comparison of the control. About 90% of the cells showed green fluorescence. The mRNA and protein in MCF-7 cells were transcription and expression of protein was significantly decreased after the transfection of MARCH6 shRNA lentivirus accompanied by a decrease in MCF-7 cell proliferation （P〈0.01）. Flow cytometry showed that the cell cycles were inhibited at the G 1 phase and the proliferation index was significantly reduced. Conclusion： Knockdown of MARCH6 gene by RNA interference inhibits the proliferation of MCF-7 cells, suggesting that the expression of MARCH6 promotes proliferation of breast cancer cells through regulation of the cell cycle.