【摘要】 目的:探讨微小RNA-141(miRNA-141)靶向Keap1调控Nrf2/ARE信号通路对乳腺癌T47D细胞活力的影响。方法:乳腺癌T47D细胞分别转染miRNA-141模拟物(mimic)和阴性对照序列(NC),作为miRNA-141组和NC组,并设立未转染空白对照组,采用real-time PCR法检测细胞中miRNA-141的含量;MTT法与荧光探针2’,7’-二氢二氯荧光素二乙酸酯(DCFH-DA)法分别检测细胞活力和活性氧簇(ROS)水平;Western blot法检测胞质接头蛋白Keap1、核因子E2相关因子2(Nrf2)、超氧化物歧化酶2(SOD2)和谷胱甘肽过氧化酶1(GPx1)的表达;双萤光素酶实验检测miRNA-141与Keap1的关系。结果:转染miRNA-141 mimic后,miRNA-141组的miRNA-141表达量明显增高,细胞活力、ROS水平和Keap1蛋白表达均下降,而细胞核Nrf2蛋白SOD2和GPx1表达升高(P<0.05);双萤光素酶实验结果显示Keap1为miRNA-141的靶基因。结论:miRNA-141可能通过靶向负调控Keap1激活Nrf2/ARE信号通路,诱导抗氧化酶的表达,以降低细胞氧化应激水平,从而抑制乳腺癌细胞的活力。更多还原

【Abstract】 AIM: To investigate the effects of microRNA-141( miRNA-141) regulating Nrf2/ARE signaling pathways by targeting Keap1 on the viability of T47 D breast cancer cells. METHODS: The breast cancer T47 D cells were transfected with miRNA-141 mimic and the negative control sequence( negative control,NC),as miRNA-141 group and NC group,respectively,and the cell without transfection was used as control group. Real-time PCR was used to detect the expression level of miRNA-141. The cell viability was measured by MTT assay. Fluorescent probe 2 ’,7 ’-dihydrodichlorofluorescein diacetate ester( DCFH-DA) was used to detect cell reactive oxygen species( ROS) level. The protein expression levels of Keap1,nuclear factor E2-related factor 2( Nrf2),superoxide dismutase 2( SOD2) and glutathione peroxidase 1( GPx1) were determined by Western blot. Dual luciferase assay was used to analyze relationship between miRNA-141 and Keap1. RESULTS: After the cells were transfected with miRNA-141 mimic,the expression of miRNA-141 was obviously higher in miRNA-141 group than that in other groups( P < 0. 05). The cell viability,ROS level and Keap1 protein expression were significantly decreased,while the Nrf2 protein in the nucleus and antioxidants SOD2 and GPx1 expression were up-regulated in miRNA-141 group. Moreover,the luciferase reporter assay demonstrated that Keap1 was the target gene of miRNA-141. CONCLUSION: miRNA-141 may negatively regulates Keap1 and activates Nrf2/ARE signaling pathways,which inhibits the viability of breast cancer cells via inducing the expression of antioxidant enzymes to reduce the oxidative stress levels of the cells.更多还原