应用RNAi敲减MARCH6基因对乳腺癌MCF-7细胞增殖及周期的影响

发布时间：2018-03-12 14:09

本文选题：MARCH　切入点：RNA干扰　出处：《中南大学学报(医学版)》2016年10期 　论文类型：期刊论文

【摘要】：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(P0.01),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。
[Abstract]:Aim: to study the effects of lentivirus-mediated RNAi on the proliferation and cell cycle of breast cancer MCF-7 cells by knockout MARCH6 gene expression. Methods: MARCH6 gene sh-#en5# lentivirus was transfected into 293T cells. The transfection efficiency was observed under fluorescence microscope, and the expression of m RNA and protein of MARCH6 gene was detected by real-time fluorescent PCR and Western blotting after lentivirus solution was infected with MARCH6 gene. The changes of cell proliferation ability were detected by MTT- Brd U cell proliferation assay. Proliferation index staining flow cytometry was used to detect the cell cycle and proliferation. Results: the vector of MARCH6sh RNA lentivirus was successfully transfected into 293T cells under fluorescence microscope and compared with the same visual field. About 80% of the cells had green fluorescence, 90% of the cells infected with MCF-7 showed green fluorescence. The expression of MARCH6 in MCF-7 cells infected with MARCH6 sh RNA lentivirus was confirmed by real-time fluorescence PCR and Western blotting. The results of Brd U assay showed that the proliferation of MCF-7 cells in MARCH6 gene knockout group decreased significantly (P 0.01). Cell cycle inhibition was observed in MCF-7 cells after MARCH6 knockout. Conclusion: after knockout of MARCH6 gene expression in breast cancer MCF-7 cells, the proliferation of MCF-7 cells was inhibited. It is suggested that MARCH6 can promote the proliferation of breast cancer cells by affecting the progress of cell cycle.
【作者单位】：中南大学湘雅医院肿瘤科;
【基金】：国家自然科学基金(81572612) 湖南省自然科学基金(2015JJ2183)~~
【分类号】：R737.9

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