miR-146a-5p靶向调控Notch2促进人晶状体上皮细胞线粒体损伤诱导的细胞凋亡

miR-146a-5p promotes cell apoptosis by inducing mitochondrial damage in human lens epithelial cells via targeting of Notch2

目的探讨miR-146a-5p在人晶状体上皮细胞线粒体损伤中的调控作用,以及miR-146a-5p靶向调控Notch2表达对细胞凋亡的影响。方法采用实时定量PCR(qRT-PCR)检测miR-146a-5p在白内障患者晶状体上皮中的表达。将miR-146a-5p模拟物转染人晶状体上皮细胞系SRA01/04,流式细胞术检测细胞凋亡。Western blotting检测凋亡相关蛋白caspase 9和caspase 12、线粒体损伤相关蛋白基质金属蛋白酶(MMP)-2和MMP-9、细胞色素c的表达。qRT-PCR检测NDUFS8、COX5b和ATP5a1 mRNA的表达。免疫荧光检测活性氧(ROS)释放。荧光素酶报告基因实验检测miR-146a-5p和Notch2的3’非翻译区靶向结合。Western blotting检测Notch2下游蛋白。结果miR-146a-5p增加了SRA01/04细胞ROS的产生,诱发凋亡。miR-146a-5p过表达通过激活caspase 9,阻断线粒体能量代谢,促进线粒体介导的细胞凋亡。Notch2被确认为miR-146a-5p的靶点。结论miR-146a-5p通过靶向调控Notch2,促进人晶状体上皮细胞线粒体损伤诱导的细胞凋亡。

Objective To investigate the regulatory role of miR-146a-5p in mitochondrial damage in human lens epithelial cells,and to explore the effect of miR-146a-5p on cell apoptosis through the targeting and regulation of Notch2 expression.Methods Realtime quantitative PCR(qRT-PCR)was used to detect the expression level of miR-146a-5p in cataract patients and non-cataract controls.miR-146a-5p mimics were transfected into human lens epithelial cell line SRA01/04,and cellular apoptosis was detected by flow cytometry.Western blotting was used to detect the expressions of the apoptosis-related proteins caspases 9 and 12,mitochondrial damagerelated proteins matrix metalloproteinase(MMP)-2 and MMP-9,and cytochrome c.The expression levels of NDUFS8,COX5b,and ATP5a1 mRNA were detected by qRT-PCR.Immunofluorescence assay was performed to detect the release of reactive oxygen species(ROS).Luciferase reporter gene assays were used to identify whether miR-146a-5p could target the 3’-untranslated region(UTR)of Notch2.The expressions of Notch2 and Hey1 were further detected by Western blotting.Results miR-146a-5p expression was upregulated in the lens epithelial samples of cataract patients.miR-146a-5p increased the production of ROS and induced apoptosis in SRA01/04 cells.miR-146a-5p mimics blocked mitochondrial energy metabolism and promoted mitochondrial dysfunction and ROS release via activation of caspases 9 and 12.We confirmed that miR-146a-5p targeted binding to the 3’-UTR of Notch2 and down-regulated the expression of Hey1 protein.Conclusion miR-146a-5p targets and regulates Notch2 to promote mitochondrial injury-induced apoptosis in human lens epithelial cells.