miR-125b通过下调MCL-1的表达提高肝癌肿瘤干细胞对多柔比星的敏感性

摘要

目的：探讨微小RNA-125b（miR-125b）是否能增强多柔比星对肝癌HepG2细胞系肿瘤干细胞（简称HepG2肿瘤干细胞）的杀伤活性并研究其机制。方法将HepG2细胞用miR-125b、多柔比星及髓细胞白血病-1（MCL-1）质粒作不同处理。采用流式细胞术检测HepG2肿瘤干细胞比例，采用噻唑蓝（MTT）法检测细胞活力，采用Annexin V染色法检测细胞凋亡率，采用JC-1染色法测定细胞线粒体膜电位，采用免疫印迹法检测目标蛋白[MCL-1、半胱氨酸天冬氨酸蛋白酶-9（caspase-9）、半胱氨酸天冬氨酸蛋白酶-3（caspase-3）]的表达。结果多柔比星单独使用能提高HepG2细胞系中肿瘤干细胞的比例，联用miR-125b后HepG2肿瘤干细胞的比例显著下降。MTT法和流式细胞术结果表明miR-125b可显著增强多柔比星对HepG2肿瘤干细胞的杀伤活性和凋亡诱导活性。JC-1染色实验结果表明miR-125b可显著增强多柔比星对HepG2肿瘤干细胞线粒体的损伤。免疫印迹法结果表明miR-125b可显著增强多柔比星依赖的caspase-9和caspase-3的活化。MCL-1质粒能明显抑制miR-125b对多柔比星的协同作用，降低二者联合作用对HepG2肿瘤干细胞活力的抑制和凋亡的诱导。结论miR-125b通过下调MCL-1的表达能提高HepG2肿瘤干细胞对多柔比星的敏感性。%Objective To investigate the role of microRNA-125b(miR-125b)increasing the killing activity of doxorubicin to hepatic cancer HepG2 stem cells and its mechanism. Methods HepG2 cells treated with miR-125b, doxorubicin and myeloid cell leukemia-1(MCL-1)plasmid. Flow cytometry was used to determine the proportions of HepG2 cells,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) method was used to determine cell vitalities,and cell apoptosis rate was determined by Annexin V staining method. Cell mitochondrial membrane potential was determined by JC-1 dyeing method,and western blotting was used to determine the target protein [MCL-1,cysteine aspartic acid specific protease-9(caspase-9) and cysteine aspartic acid specific protease-3 (caspase-3)]. Results Single treatment of doxorubicin increased the proportion of HepG2 cells,however,the combined treatment with doxorubicin and miR-125b decreased it. The results of MTT method and flow cytometry showed that miR-125b could enrich the killing activity and cell apoptosis of doxorubicin to HepG2 cells. The results of JC-1 dyeing method showed that miR-125b enhanced the damage of mitochondria induced by doxorubicin. The results of western blotting indicated that miR-125b increased the activation of caspase-9 and caspase-3 in doxorubicin-treated HepG2 cells. MCL-1 plasmid impaired the synergistic effect of miR-125b on doxorubicin-induced cell death and apoptosis in HepG2 cells. Conclusions miR-125b increased the susceptibility of HepG2 cells to doxorubicin through down-regulating the expression of MCL-1.