定量沉默c-Met基因表达对乳腺癌细胞MDA-MB-231增殖和化疗敏感性的影响

Effect of quantitative silencing of c-Met gene expression on the proliferation and chemosensitivity of breast cancer cell line MDA-MB-231

目的探讨定量沉默c-Met基因表达对人乳腺癌细胞MDA-MB-231增殖、表阿霉素化疗敏感性的影响及其可能机制。方法设计3条针对c-Met基因不同位点短发夹RNA(shRNA)片段,瞬时转染MDA-MB-231细胞,挑选出沉默效率最佳的shRNA,并设定scramble为阴性对照组,MDA-MB-231细胞为空白对照组。将沉默效率最佳的TA-shRNA连接到PSD400慢病毒载体中进行病毒包装,收集病毒液并感染MDA-MB-231细胞,利用嘌呤霉素筛选出稳定表达针对c-Met的shRNA细胞系(PSD400-c-Met-shRNA-MDA-MB-231)及阴性对照组细胞(PSD400-scramble-MDA-MB-231)。利用多西环素(DOX)诱导稳定细胞系,Western blot检测稳定细胞系c-Met蛋白表达水平,噻唑蓝(MTT)法检测敲低c-Met后对稳定细胞系增殖的影响。将诱导的稳定细胞系加入不同浓度表阿霉素,MTT法检测不同浓度表阿霉素作用对稳定细胞系存活率的改变,计算出药物半数抑制浓度(IC_(50));流式细胞技术检测细胞凋亡情况;Western blot检测凋亡相关蛋白多聚二磷酸腺苷(ADP)-核糖聚合酶(PARP)、天冬氨酸特异性半胱氨酸蛋白酶-3(caspase-3)的表达。结果稳定细胞系PSD400-c-Met-shRNA-MDA-MB-231经DOX诱导后细胞生长受到明显抑制,且加入不同浓度表阿霉素处理细胞后,细胞的存活率和IC_(50)均明显降低(P<0.05);同时G_(0)/G_(1)期细胞百分比明显升高,S期、G_(2)/M期细胞百分比明显降低(P<0.05)。Western blot检测凋亡相关蛋白PARP和caspase-3上调明显。结论定量沉默c-Met基因可以抑制MDA-MB-231细胞的增殖,促进细胞凋亡,并提高了MDA-MB-231细胞的化疗敏感性,靶向c-Met基因可能是治疗乳腺癌的有效方法。

Objective To investigate the effect of quantitative silencing of c-Met gene expression on the proliferation of human breast cancer cell line MDA-MB-231 and the chemosensitivity of epirubicin(EPI)and its possible mechanism.Methods Three short hairpin RNA(shRNA)fragments targeting different sites of the c-Met gene were designed and transiently transfected into MDA-MB-231 cells.The shRNA with the best silencing efficiency was selected,scramble was set as the negative control group,and MDA-MB-231 cells were set as the blank control group.The TA-shRNA with the best silencing efficiency was connected to the PSD400 lentiviral vector for virus packaging,the virus liquid was collected and infected with MDA-MB-231 cells,and puromycin was used to screen out the cell lines that stably express shRNA against c-Met(PSD400-c-Met-shRNA-MDA-MB-231)and the negative control cells(PSD400-scramble-MDA-MB-231).The stable cell lines were induced by doxycycline(DOX),the protein expression level of c-Met in stable cell lines was detected by Western blot,and the effect of knockdown of c-Met on the proliferation of stable cell lines was detected by thiazolylblue(MTT)assay.The induced stable cell lines were added with different concentrations of epirubicin,the changes in the survival rate of stable cell lines caused by the effect of different concentrations of epirubicin were detected by MTT assay,and the median inhibitory concentration(IC_(50))of the drug was calculated.Flow cytometry was used to detect cell apoptosis.The expression levels of apoptosis-related proteins,adenosine diphosphate-ribose polymerase(ADP-PARP)and caspase-3,were detected by Western blot.Results The growth of stable cell line PSD400-c-Met-shRNA-MDA-MB-231 was significantly inhibited by DOX induction,and the cell viability and IC_(50) were significantly decreased after treated with different concentrations of epirubicin(P<0.05).At the same time,the percentage of cells in G_(0)/G_(1) phase was significantly increased,and the percentage of cells in S phase and G_(2)/M phase was significantly decreased(P<0.05).The results of Western blot showed that apoptosis-related proteins,PARP and caspase-3,were significantly up-regulated.Conclusion Quantitative silencing of c-Met gene can inhibit the proliferation of MDA-MB-231 cells,promote cell apoptosis,and improve the chemosensitivity of MDA-MB-231 cells.Targeting c-Met gene may be an effective method for the treatment of breast cancer.