Med19基因敲减增加乳腺癌细胞化疗敏感性及其可能的机制

Med19 knockdown promotes chemosensitivity of breast cancer cells and its possible mechanism

目的:研究中介体(mediator,Med)19对乳腺癌化疗敏感性的影响并分析其分子机制。方法:选用多柔比星(adriamycin,ADM)耐药的人乳腺癌细胞MCF-7/ADM和亲本细胞MCF-7(NC组),采用慢病毒载体介导RNA干扰方法构建Med19稳定低表达的MCF-7/ADM与MCF-7细胞株(KD组),并用Real-time PCR和Western blotting方法验证干扰效果。CCK-8法检测慢病毒介导的Med19敲减前后两种细胞对ADM、顺铂(cisplatin,DDP)和紫杉醇(taxinol,TAX)药物敏感性的变化。Real-time PCR和Western blotting检测Med19敲减对多药耐药基因1(multidrug resistance 1,MDR1)和细胞凋亡基因Bcl2、Bax及Caspase-3、active Caspase-3的表达的影响。流式细胞术检测敲减Med19及ADM处理对细胞凋亡的影响。结果:与MCF-7相比,MCF-7/ADM细胞对ADM、DDP和TAX均具有耐药性。成功构建Med19稳定低表达的MCF-7/ADM与MCF-7细胞株,并且其对ADM、DDP、TAX的敏感性增加,药物作用IC_(50)显著降低(均P<0.05)。MCF-7/ADM细胞Med19 mRNA和蛋白表达显著高于MCF-7细胞,Med19的敲减可降低MCF-7/ADM细胞中MDR1 mRNA与蛋白表达水平(均P<0.05)并可增加MCF-7/ADM及MCF-7细胞中凋亡相关active Caspase-3、Bax的蛋白表达,降低Bcl2的蛋白表达(均P<0.05)。此外,与NC及NC+ADM组相比,KD组及KD+ADM组凋亡水平显著增加(均P<0.05)。结论:Med19高表达参与乳腺癌化疗耐药,其机制可能与Med19调节MDR1的表达并影响细胞凋亡有关。

Objective：To explore the effect and molecular mechanism of mediator 19 （Med19） on drug sensitivity of breast cancer cells. Methods： Human breast cancer cell line MCF-7/ADM （adriamycin resistant） and it’s parental cell line MCF-7（NC groups） were selected. Lentivirus-mediated RNA-interference was used to construct MCF-7/ADM and MCF-7 cell lines that stably expressing low Med19（KD groups）, and the infection efficiency was detected by Real-time PCR and Western blotting assays. The change of drug-susceptibility to ADM, cisplatin （DDP） and taxinol （TAX） were examined with CCK-8 assay before and after Med19 knockdown. The effects of Med19 knockdown on the expressions of multidrug resistance gene 1（MDR1）, Bcl2, Bax,Caspase-3 and active caspase-3 in breast cancer cells were detected by RT-PCR and Western Blotting assays, respectively. Effect of Med19 down-regulation and ADM treatment on apoptosis rates of MCF-7 and MCF-7/ADM cells were detected with flow cytometry assay. Results： Compared with MCF-7 cells, MCF-7/ADM cells exhibited drug resistance to ADM, DDP and TAX. MCF-7/ADM and MCF-7 cell lines that stably expressing low Med19level were successfully established; their drug-susceptibilities to ADM, DDP, TAX were increased, and the IC50 values of these three cytotoxic agents deceased obviously （all P〈0.05）. The mRNA and protein expressions of Med19in MCF-7/ADM cells were significantly higher than those in MCF-7 cells, however, Med19knockdown significantly inhibited the expressions of MDR1mRNA and protein in MCF-7/ADM cells （all P〈0.05）; in addition, it significantly increased the expressions of active caspase-3 and Bax protein in both MCF-7/ADM and MCF-7 cells （all P〈0.05） and decreased the expression of Bcl2 protein （all P〈0.05）. Besides, compared with NC or NC＋ADM group, the apoptosis rate of the cells significantly increased in KD group or KD＋ADM group （all P〈0.05）. Conclusions： High Med19expression plays a particularly important role in chemoresistance of breast cancer, and the mechanism might correlate with Med19regulating MDR1 expression and affecting apoptosis related genes.