microRNA-125b通过调控Nrf2/Keap1信号通路影响光感受器细胞氧化应激  

Regulatory role of microRNA-125b in oxidative stress of photoreceptor cells via the Nrf2/Keap1 signaling pathway

作　　者：刘金霞 王钰池 郭卓 赵江月[1] 孔珺[1] 秦宇[1] LIU Jinxia;WANG Yuchi;GUO Zhuo;ZHAO Jiangyue;KONG Jun;QIN Yu(Department of Ophthalmology,The Fourth Affiliated Hospital of China Medical University,Eye Hospital of China Medical University,Key Lens Research Laboratory of Liaoning Province,Shenyang 110005,China)

机构地区：[1]中国医科大学附属第四医院眼科,中国医科大学眼科医院,辽宁省晶状体学重点实验室,沈阳110005

出　　处：《中国医科大学学报》2021年第11期976-980,985,共6页Journal of China Medical University

基　　金：国家自然科学基金青年基金(81600717);辽宁省自然科学基金(201602851)。

摘　　要：目的研究在H_(2)O_(2)诱导的光感受器细胞氧化应激模型中microRNA-125b(miR-125b)调控核因子E2相关因子2(Nrf2)/Kelch样环氧氯丙烷相关蛋白1(Keap1)信号通路的作用机制,探讨其在干性年龄相关性黄斑变性(AMD)发病过程中的作用。方法利用H_(2)O_(2)(800μmol/L)构建小鼠视锥细胞系661W细胞氧化应激模型,用实时荧光定量PCR(q PCR)检测mi R-125b及Keap1/Nrf2/HO-1 mRNA的表达。分别转染miR-125b模拟物、模拟物对照、抑制物、抑制物对照至661W细胞中,调控细胞miR-125b的表达,48h后用qPCR在661W细胞氧化应激模型中检测miR-125b及Keap1/Nrf2/HO-1 mRNA的表达。用Western blotting检测Keap1蛋白的表达。结果与对照组相比,miR-125b的表达在661W细胞氧化应激模型中显著降低,Nrf2及其下游基因HO-1 mRNA表达显著升高,Keap1 mRNA表达明显降低;与转染miR-125b模拟物对照组相比,转染miR-125b模拟物组Nrf2、HO-1与miR-125b mRNA表达显著升高,Keap1表达在mRNA水平无明显变化,在蛋白水平显著降低;与转染miR-125b抑制物对照组相比,转染miR-125b抑制物组Nrf2、HO-1与miR-125b mRNA表达显著降低,Keap1 mRNA表达水平无明显变化,蛋白表达显著增加,差异均有统计学意义(P<0.05)。结论miR-125b可能通过调控Nrf2/Keap1信号通路影响光感受器细胞氧化应激反应,调控干性AMD的发生,因而可能成为干性AMD治疗的新靶点。Objective To investigate the regulating mechanism of microRNA-125 b(miR-125 b)in photoreceptor cells via the Nrf2/Keap1 signaling pathway using an H_(2)O_(2)-induced oxidative stress model.Methods The oxidative stress model was established using the mouse cone cell line 661 W treated with H_(2)O_(2)(800μmol/L)for 6 h.Expression of miR-125 b and Keap1/Nrf2/HO-1 was detected using real-time quantitative PCR(qPCR).miR-125 b mimic and mimic control and miR-125 b inhibitor and inhibitor control were transfected into the treated 661 W cells in serum-free Opti-MEM using LipofectamineTM RNAiMAX.miR-125 b expression,48 hours post-transfection,in 661 W cells was up-and downegulated,respectively.Expression of Keap1 protein was detected using Western blotting.Results Compared to the control group,expression of miR-125 b decreased significantly,that of Nrf2 and its downstream gene HO-1 increased significantly,and that of Keap1 decreased significantly in the H_(2)O_(2)-treated group.Furthermore,after transfection with miR-125 b mimic,the mRNA expression of Nrf2,HO-1,and miR-125 b increased significantly.However,Keap1 mRNA expression showed no significant change,whereas Keap1 protein expression decreased significantly.Conversely,after transfection with mi R-125 b inhibitor,m RNA expression of Nrf2,HO-1,and mi R-125 b was significantly downregulated and that of Keap1 showed no significant change,whereas Keap1 protein expression level significantly increased.All differences were significant(P<0.05).Conclusion The miR-125 b/Keap1/Nrf2/HO-1 axis is a potential regulatory mechanism in pathogenesis and treatment of dry age-related macular degeneration that witnesses severe oxidative stress and adversely damaged photoreceptor cells.

关 键 词：microRNA-125b 核因子E2相关因子 氧化应激 干性年龄相关性黄斑变性 

分 类 号：R774.1[医药卫生—眼科]