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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 investigate the effect of mediator (Med) 19 on the chemosensitivity of breast cancer and analyze its molecular mechanism. Methods: Adriamycin resistant human breast cancer cells MCF-7 / ADM and parental cells MCF-7 (NC group) were selected. Lentiviral vector-mediated RNAi was used to construct stable and low expression of Med19 MCF-7 / ADM and MCF-7 cell lines (KD group). Real-time PCR and Western blotting were used to verify the interference effect. CCK-8 was used to detect the changes of lentivirus-mediated drug sensitivity to ADM, cisplatin (DDP) and taxol (TAX) before and after Med19 knockdown. Real-time PCR and Western blotting were used to detect the effect of Med19 knockdown on the expression of multidrug resistance 1 (MDR1), apoptosis-related genes Bcl2, Bax, Caspase-3 and active Caspase-3. Flow cytometry was used to detect the effect of knockdown of Med19 and ADM treatment on apoptosis. Results: Compared with MCF-7, MCF-7 / ADM cells were resistant to ADM, DDP and TAX. The stable and low expression of Med19 in MCF-7 / ADM and MCF-7 cells was successfully established, and its sensitivity to ADM, DDP and TAX was increased. The IC 50 of drug was significantly reduced (all P <0.05). The expression of Med19 mRNA and protein in MCF-7 / ADM cells was significantly higher than that in MCF-7 cells. Med19 knockdown could decrease the expression of MDR1 mRNA and protein in MCF-7 / ADM cells (all P <0.05) / ADM and MCF-7 cells, and decreased the expressions of active Caspase-3 and Bax protein (all P <0.05). In addition, the apoptotic levels in KD and KD + ADM groups were significantly higher than those in NC and NC + ADM groups (all P <0.05). Conclusion: The high expression of Med19 is involved in chemoresistance in breast cancer. The mechanism may be related to the regulation of MDR1 expression by Med19 and the effect on apoptosis.