摘要： 目的 研究在H₂O₂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H₂O₂ （800 μmol/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 （qPCR）检测miR-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，48 h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。

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

刘金霞, 王钰池, 郭卓, 赵江月, 孔珺, 秦宇

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

收稿日期:2021-01-27出版日期:2021-11-30发布日期:2021-11-04
通讯作者:秦宇E-mail:qinyubb@126.com
作者简介:刘金霞(1995-),女,硕士研究生.
基金资助:国家自然科学基金青年基金（81600717）；辽宁省自然科学基金（201602851）


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
Abstract: Objective To investigate the regulating mechanism of microRNA-125b (miR-125b) in photoreceptor cells via the Nrf2/Keap1 signaling pathway using an H₂O₂-induced oxidative stress model. Methods The oxidative stress model was established using the mouse cone cell line 661W treated with H₂O₂ (800 μmol/L) for 6 h. Expression of miR-125b and Keap1/Nrf2/HO-1 was detected using real-time quantitative PCR (qPCR). miR-125b mimic and mimic control and miR-125b inhibitor and inhibitor control were transfected into the treated 661W cells in serum-free Opti-MEM using Lipofectamine^TM RNAiMAX. miR-125b expression, 48 hours post-transfection, in 661W cells was up- and downegulated, respectively. Expression of Keap1 protein was detected using Western blotting. Results Compared to the control group, expression of miR-125b decreased significantly, that of Nrf2 and its downstream gene HO-1 increased significantly, and that of Keap1 decreased significantly in the H₂O₂-treated group. Furthermore, after transfection with miR-125b mimic, the mRNA expression of Nrf2, HO-1, and miR-125b increased significantly. However, Keap1 mRNA expression showed no significant change, whereas Keap1 protein expression decreased significantly. Conversely, after transfection with miR-125b inhibitor, mRNA expression of Nrf2, HO-1, and miR-125b 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-125b/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.
Key words: microRNA-125b, nuclear factor-erythroid2-related factor 2, oxidative stress, dry age-related macular degeneration
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