miR-34a-5p通过调节Nrf2-Keap1信号通路介导年龄相关性白内障氧化应激的实验研究

目的探讨miR-34a-5p通过调节Nrf2-Keap1信号通路介导年龄相关性白内障的氧化应激。方法检测年龄相关性白内障患者和正常透明晶状体前囊组织及人晶状体上皮细胞氧化应激模型中miR-34a-5p和预测靶基因Nrf2表达及人晶状体上皮细胞内活性氧(reactive oxygen species,ROS)活性。将miR-34a-5p模拟物、模拟物对照、miR-34a-5p抑制剂和抑制剂对照分别转染人晶状体上皮细胞,然后用400μmol·L^-1 H2O2作用8 h后检测miR-34a-5p、Nrf2 mRNA和Nrf2蛋白表达,并检测人晶状体上皮细胞增殖活性。结果正常晶状体前囊组织中miR-34a-5p的相对表达量为1.03±0.29,Nrf2 mRNA的相对表达量为1.31±0.39;年龄相关性白内障组织中miR-34a-5p的相对表达量为2.69±0.99,Nrf2 mRNA的相对表达量为0.64±0.25。与正常组织相比,年龄相关性白内障晶状体组织中miR-34a-5p表达显著升高,Nrf2表达显著降低。Nrf2蛋白在年龄相关性白内障晶状体组织中表达也显著降低(均为P<0.001)。在人晶状体上皮细胞氧化应激模型中,miR-34a-5p表达显著升高,靶基因Nrf2表达显著降低,内源性ROS水平显著升高(均为P<0.001)。miR-34a-5p模拟物转染人晶状体上皮细胞后,miR-34a-5p表达显著升高,Nrf2表达显著降低,内源性ROS水平显著升高,细胞增殖活性显著降低(均为P<0.001),然而,miR-34a-5p抑制剂转染人晶状体上皮细胞后,miR-34a-5p表达显著降低,Nrf2表达显著升高,内源性ROS水平显著降低,细胞增殖活性显著升高(均为P<0.001)。双荧光素酶报告分析证实,Nrf2是miR-34a-5p的直接靶点。结论 MiR-34a-5p通过调节Nrf2-Keap1信号通路增加晶状体上皮细胞氧化应激,抑制晶状体上皮细胞的增殖,从而参与年龄相关性白内障的发生发展过程。

MiR-34a-5p mediates oxidative stress in age-related cataract via regulating Nrf2-Keap1 signal pathway

Objective　To investigate miR-34a-5p mediates oxidative stress in age-related cataract via regulating Nrf2-Keap1 signal pathway.Methods　The miR-34a-5p,the target gene(Nrf2) and intracellular reactive oxygen species (ROS) were detected in the anterior capsule of clear lens and the oxidative stress model of human lens epithelial cells,respectively.Then,miR-34a-5p mimics,mimics control,miR-34a-5p inhibitors and inhibitors control were transfected into human lens epithelial cells,respectively,the cells were exposed to 400 μmol·L^-1 H₂O₂ for 8h,the miR-34a-5p,target gene mRNA and protein was detected and the proliferation activity of cells was also detected.Results　The relative expression of miR-34a-5p and Nrf2 in normal tissues was 1.03±0.29 and 1.31±0.39,respectively.The relative expression of miR-34a-5p and Nrf2 in age-related cataract tissue was 2.69 +0.99 and 0.64 +0.25,respectively.Compared with the normal group,the expression of miR-34a-5p in lens tissue of age-related cataract was significantly increased,the expression of Nrf2 mRNA was significantly decreased,and the expression of Nrf2 protein in lens tissue of age-related cataract was also significantly decreased (all P<0.001).In the oxidative stress model of human lens epithelial cells,the level of miR-34a-5p was significantly increased,target gene (Nrf2) was significantly decreased and endogenous ROS were significantly increased(all P<0.001).For miR-34a-5p mimics,the miR-34a-5p was significantly increased,the Nrf2 was significantly decreased,the level of endogenous ROS was significantly increased,and the cell proliferation activity was significantly decreased (all P<0.001),while for miR-34a-5p inhibitors,the miR-34a-5p was significantly decreased,the Nrf2 was significantly increased,the level of endogenous ROS was significantly decreased,and the cell proliferation activity was significantly increased (all P<0.001).Finally,the dual luciferase reporter assay confirmed that Nrf2 was a direct target of miR-34a-5p.Conclusion　miR-34a-5p has the ability to enhance oxidative stress in human lens epithelial cells,restrains the proliferation of human lens epithelial cells by regulating the Nrf2-Keap1 signal pathway,thus participating in the process of age-related cataract.