MiR-34a-5p Negatively Regulates Oxidative Stress on Lens Epithelial Cells by Silencing GPX3 – A Novel Target

MiR-34a-5p is reported to be related with age-related nuclear cataract. This study investigated the mechanism of miR-34a-5p in the regulation of oxidative stress on lens epithelial cells. The three candidate miRNAs were screened by CCK-8 assays after transfection of mimics or inhibitor in H₂O₂-treated HLE-B3 cells. The apoptosis, ROS level and GPX activity of HLE-B3 cells transfected with miR-34a-5p mimics or inhibitor were analysed by flow cytometry, cellular ROS and GPX activity test. The target genes of miR-34a-5p were predicted by proteomic and bioinformatic analysis. The relationship between miR-34a-5p and GPX3 were internally validated by qRT-PCR and Western blot and externally verified by dual-luciferase reporter assay. The effect of miR-34a-5p-GPX3 axis on regulation of oxidative stress in HLE-B3 cells were conducted by overexpression of GPX3 and tested by flow cytometry analysis, cellular ROS and GPX detection. The viability of H₂O₂-treated HLE-B3 cells were weakened by up-regulated miR-34a-5p. Cell apoptosis and oxidative damage were also induced by overexpression of miR-34a-5p. GPX3 and SRC were identified as target genes of miR-34a-5p by combined analysis of proteomic and bioinformatics, while GPX3 was selected for further research for its connection with anti-oxidation. Western blot and qRT-PCR tests proved that GPX3 is negatively regulated by miR-34a-5p. Dual-luciferase reporter assay verified that GPX3 is the direct target of miR-34a-5p. The increased oxidative stress induced by transfection of miR-34a-5p mimics in H₂O₂-treated HLE-B3 cells was attenuated by overexpression of GPX3. MiR-34a-5p is a negative regulator of oxidative stress on lens epithelial cells and the mechanism is by silencing the expression of GPX3. These data suggest that miR-34a-5p may be a potential novel therapeutic target for the prevention and treatment of age-related cataract.

已有研究证实，miR-34a-5p与年龄相关性核性白内障（age-related nuclear cataract）存在密切关联。本研究旨在探讨miR-34a-5p在氧化应激调控晶状体上皮细胞过程中的作用机制。研究人员在经过氧化氢（H₂O₂）处理的HLE-B3细胞中转染miR-34a-5p模拟物（mimics）或抑制剂（inhibitor）后，通过CCK-8实验筛选出3种候选miRNA。随后，分别采用流式细胞术、细胞活性氧（ROS，Reactive Oxygen Species）检测法及谷胱甘肽过氧化物酶（GPX，Glutathione Peroxidase）活性检测，分析转染miR-34a-5p模拟物或抑制剂的HLE-B3细胞的凋亡水平、ROS含量及GPX活性。通过蛋白质组学与生物信息学分析，预测miR-34a-5p的靶基因；采用实时定量荧光PCR（qRT-PCR，quantitative real-time PCR）与蛋白质免疫印迹实验（Western blot）对内源性miR-34a-5p与谷胱甘肽过氧化物酶3（GPX3）的靶向关系进行内部验证，并通过双荧光素酶报告基因实验完成外部验证。为探究miR-34a-5p-GPX3轴对HLE-B3细胞氧化应激的调控作用，本研究通过过表达GPX3构建干预体系，并采用流式细胞术、细胞ROS检测及GPX活性检测进行功能验证。实验结果显示，上调miR-34a-5p的表达会削弱经H₂O₂处理的HLE-B3细胞的存活率，同时诱导细胞凋亡与氧化损伤。联合蛋白质组学与生物信息学分析，鉴定出GPX3与SRC为miR-34a-5p的靶基因；鉴于GPX3与抗氧化功能的相关性，选取其开展后续深入研究。蛋白质免疫印迹与qRT-PCR实验证实，miR-34a-5p对GPX3的表达具有负调控作用；双荧光素酶报告基因实验进一步验证GPX3是miR-34a-5p的直接靶基因。在经H₂O₂处理的HLE-B3细胞中，转染miR-34a-5p模拟物所诱导的氧化应激升高，可通过过表达GPX3得到显著缓解。综上，miR-34a-5p是晶状体上皮细胞氧化应激的负调控因子，其作用机制为沉默GPX3的基因表达。本研究结果提示，miR-34a-5p有望成为年龄相关性白内障防治的潜在新型治疗靶点。