慢病毒介导的靶向c-Met可诱导shRNA稳定乳腺癌MDA-MB-231细胞系的构建及生物活性鉴定

Construction and Biological Activity Identification of Breast Cancer MDA-MB-231 Stable Cell Line with Lentivirus-Mediated shRNA Targeting c-Met

目的:探讨适当敲除c-Met后对乳腺癌MDA-MB-231细胞增殖的影响。方法:设计3条针对c-Met基因不同位点的短发夹RNA(shRNA)片段,将其连接到TA载体上,瞬时转染MDA-MB-231细胞,采用RT-PCR、Western印迹检测c-Met mRNA及蛋白表达情况,挑选沉默效率最佳的shRNA。以scramble为阴性对照,MDA-MB-231细胞为空白对照,挑选沉默效率最佳的TA-shRNA,并将其与pSD400慢病毒载体连接,利用293T细胞进行慢病毒组装,收集病毒液并感染MDA-MB-231细胞,将嘌呤霉素与MDA-MB-231细胞共培养,筛选稳定表达shRNA的细胞株。利用多西环素对稳定细胞系进行诱导,Western印迹检测稳定细胞系c-Met蛋白的表达量,MTT法检测敲低c-Met对稳定细胞系增殖的影响。结果:测序结果表明模板序列与设计序列正确,并筛选出沉默效率最高的shRNA为shRNA1,慢病毒包装后筛选出稳定细胞株,适当敲除MDA-MB-231细胞c-Met基因后细胞的增殖明显低于对照组。结论:构建了针对c-Met基因可诱导shRNA的乳腺癌稳定细胞系,为探讨适当敲除c-Met基因后乳腺癌细胞对化疗、放疗敏感性检测奠定了基础。

Objective:To construct the stable cell line using the virus with the plasmid expressing short hairpin RNA(shRNA)against human c-Met gene and discuss the influences of appropriate c-Met knockdown on breast cancer MDA-MB-231 cells.Methods:Design 3 pairs of shRNA targeting different sites of c-Met gene,then insert into TA plasmid.Select the cells with best knockdown efficiency using Western blotting and RT-PCR after transiently transfecting the plasmid into MDA-MB-231 cells.The scramble and empty MDA-MB-231 cells were conducted as negative control and bank control respectively.Put the TA-shRNA of the shRNA of best efficiency insert into pSD400 Lentivirus vector,and package the virus.Then collect the virus to infect MDA-MB-231 cells.24 h later,use puromycin to select the cells stably expressing c-Met shRNA.Induce the stable cell line with doxycycline to detect the expression level of c-Met protein using Western blotting and the influences of c-Met knockdown on MDA-MB-231 cells multiplication.Results:The template and the design sequences were confirmed by sequencing.The potency of knock-down the shRNA1 was the greatest.After the lentivirus was packaged,the stable cell line was screened.The proliferation of MDA-MB-231 cells with c-Met gene knock-down was significantly lower than that of control group.Conclusion:A stable breast cancer cell line that can regulate shRNA breast cancer targeting c-Met gene was successfully constructed,which laid a foundation for the next step to explore the effect of c-Met gene knock-down on the biological behavior of breast cancer cells.