MicroRNA-140在乳腺癌细胞中对Mrf2及其下游抗氧化基因的表达调控  

The regulation of Nrf2 and its downstream antioxidant gene expression by microRNA-140 in breast cancer cells

作　　者：于影[1] 李豪杰[1] 温传俊[1] 

机构地区：[1]南京师范大学生命科学学院,210023

出　　处：《国际免疫学杂志》2013年第4期303-309,共7页International Journal of Immunology

摘　　要：目的本研究旨在揭示microRNA在乳腺癌细胞中对哪表达的影响。方法利用生物信意学方法预测miR-140与Ⅳ啦的结合位点，然后采用双荧光报告系统检测miR-140对Nrf23’UTR及启动子上的8×ARE区域活性的影响；同时以实时荧光定量PCR法和Westernbloting法检测过表达miR．140和加药刺激前后细胞中Nrf2及其下游基因的mRNA和蛋白的表达变化；最后，四甲基偶氮唑蓝（MTY）法检测转染miR-140后，对于不同浓度H2O2处理的细胞，观察其生长能力及对氧化应激敏感性的变化。结果过表达miR．140能够显著抑制Nrf啦5’UTR区域的表达（P=0．007）和启动子上ARE区域的活性（P=0．01）；在过表达miR-140的对照组和加药组细胞中，Ⅳ以及其下游基因mRNA和蛋白水平均被明显抑制（均P〈0．05）；MTT实验结果显示转染miR-140细胞活力受到抑制，同时再使用不同浓度的H2O2刺激细胞后，细胞对氧化应激的敏感性增加（P〈0．01）。结论Ⅳ啦可能作为miR-140的一个新型靶基因，miR-140能够通过抑制Ⅳ犯，进而影响其下游一系列抗氧化基因的表达。Objective To dissect the effects of microRNA on Nrf2 expression in breast cancer cells. Methods Predicted the directly binding sites between miR-140 and Nrf2 by using bioinformatics analysis （miRbase and Target Scan）. After that,we performed dual-luciferase reporter assay to analyse the effects of miR-140 on the expression of Nrf2 3＇UTR and the 8 x ARE luciferase activity. After Pre-treatment with/without mir-140 and drugs, cells were identified Nrf2 and downstream genes mRNA and protein levels lysed in trizol and RIPA-buffer respectively by RT-qPCR and Western blot . Cell viability and the oxidative stress sensitivity were determined by MT＇F assay after miR-140 overexpression/different concentrations of H202. Results Tran- sient transfected with miR-140 suppressed both Nrf2 3 ＇UTR expression（ P = 0.007 ） and ARE luciferase activity （P =0. 01 ）. Furthermore, transfected with miR-140 inhibited Nrf2 and its downstream genes expression in both mRNA and protein levels in breast cancer cells（P 〈 0.05）. The last but not the least, MTF assay showed cell viablity was inhibited after transfected with miR-140, and the oxidative Stress sensitivity was increased while stimulated cells with different concentrations of H2 02 （P 〈 0.01 ）. Conclusion Nrf2 may serve as a new target gene of miR-140, and miR-140 repressed Nrf2 expression and inhibited its downstream antioxidant path- ways

关 键 词：miR-140 NRF2 ARE 细胞生长 氧化应激 

分 类 号：R737.9[医药卫生—肿瘤]