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。
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.
作者
常景芝
陈剑
芦琨
郭亚莉
沈永杰
李宜川
张善锋
CHANG Jing-zhi;CHEN Jian;LU Kun;GUO Ya-li;SHEN Yong-jie;LI Yi-chuan;ZHANG Shan-feng(Department of Biochemistry and Molecular Biology,Shangqiu Medical College,Shangqiu 476100,China;School ofBasic Medical Sciences,Zhengzhou University,Zhengzhou 450001,China.)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2018年第7期1245-1249,共5页
Chinese Journal of Pathophysiology
基金
2015年河南省重点科技攻关项目(No.152102310235)
