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

doi:10.3969/j.issn.1673-8640.2016.011.013

摘要/Abstract

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。

关键词: 微小RNA-125b, 多柔比星, 肝癌, 肿瘤干细胞, 髓细胞白血病基因1, 细胞凋亡

Abstract:

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.

Key words: MicroRNA-125b, Doxorubicin, Hepatic cancer, Cancer stem cells, Myeloid cell leukemia-1, Cell apoptosis

中图分类号:

R446.1

洪东承, 张本宏, 龚彬彬. 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[J]. 检验医学, 2016, 31(11): 981-986.

HONG Dongcheng, ZHANG Benhong, GONG Binbin. MiR-125b increasing the susceptibility of hepatic cancer stem cells to doxorubicin through down-regulating the expression of MCL-1[J]. Laboratory Medicine, 2016, 31(11): 981-986.

图/表 8

图1 miR-125b和多柔比星处理对肝癌HepG2细胞系中肿瘤干细胞种群比例的影响

图2 HepG2肿瘤干细胞的分选效率

图3 各组HepG2肿瘤干细胞细胞活力抑制率的比较

图4 各组HepG2肿瘤干细胞凋亡实验结果

表1 各组HepG2肿瘤干细胞线粒体膜电位caspase-9、caspase-3相对表达水平的比较（x±s）

组别	相对线粒体膜电位	caspase-9	caspase-3
对照组	1.00±0.05	0.01±0.01	0.01±0.01
多柔比星组	0.82±0.04^*#	0.13±0.02^*#	0.15±0.02^*#
miR-125b组	0.94±0.05	0.01±0.01	0.01±0.01
多柔比星+ miR-125b组	0.34±0.02^*#△	0.55±0.04^*#△	0.61±0.05^*#△

图5 各组HepG2肿瘤干细胞活化caspase-9和caspase-3的表达水平

表3 各组HepG2肿瘤干细胞MCL-1相对表达水平及活力抑制率的比较（x±s）

组别	MCL-1相对表达水平	细胞活力抑制率（%）
对照组	0.83±0.05	0
多柔比星组	0.85±0.06	13.5±0.8^*
miR-125b组	0.24±0.02^*#	6.3±0.3^*#
多柔比星+miR-125b组	0.23±0.02^*#	67.5±3.7^*#
多柔比星+miR-125b+ MCL-1质粒组	0.91±0.06^△▲	19.3±1.0^*#△▲

图6 各组HepG2肿瘤干细胞中MCL-1的表达

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