miR-125b在小鼠白内障模型与人晶状体上皮细胞凋亡模型中的调控作用

Investigations on the regulatory role of microRNA-125b in a mouse cataract model and human lens epithelial cell apoptosis model

目的研究在紫外线诱导的小鼠白内障模型与人晶状体上皮细胞凋亡模型中微小RNA-125b(miR-125b)靶向调控BAK1的作用机制。方法利用LipofectamineTM RNAiMAX分别转染miR-125b模拟物(mimic)、模拟物阴性对照(mimic control)、miR-125b抑制物(inhibitor)、抑制物阴性对照(inhibitor control)至人晶状体上皮细胞系SRA01/04,分别上调和下调细胞中miR-125b的表达,48 h后用紫外线UVB(360μW/cm2)照射25 min,构建细胞凋亡模型。用UVB(360μW/cm2)照射小鼠左眼,制作白内障模型,收集小鼠晶状体囊膜。用实时qPCR验证转染效率,并检测miR-125b及其预测靶基因BAK1 mRNA的表达。用Western blotting检测BAK1蛋白的表达,用TUNEL凋亡检测法检测人晶状体上皮细胞凋亡率。结果与正常人晶状体上皮细胞比较,人晶状体上皮细胞凋亡模型中miR-125b的表达显著降低,BAK1表达显著升高,细胞凋亡增加;与转染miR-125b mimic control组相比,转染miR-125b mimic组miR-125b的表达显著增高,BAK1表达显著降低,细胞凋亡减少;与转染miR-125b inhibitor control组相比,转染miR-125b inhibitor组miR-125b的表达显著降低,BAK1表达显著升高,细胞凋亡增加。与未照射组相比,紫外线UVB照射组小鼠晶状体囊膜组织中miR-125b的表达显著降低,BAK1表达显著升高,细胞凋亡增加。结论miRNA-125b可能通过靶向调控BAK1影响人晶状体上皮细胞凋亡,从而调控白内障发病,可能成为非手术靶向治疗白内障的新方法。

Objective To investigate the mechanism of targeting regulatory BAK1 protein by microRNA-125b(miR-125b)in an UVinduced mouse cataract model and a human lens epithelial cell apoptosis model.Methods LipofectamineTM RNAiMAX was used to transfect either miR-125b mimics or control RNAs,along with miR-125b inhibitor molecules and inhibitor controls to human lens epithelial cell line SRA01/04,either to up-regulate or down-regulate expression levels of miR-125b,respectively.After 48 h,ultraviolet UVB light(360μW/cm2)was used to irradiate for 25 min to establish an apoptosis model.The cataract model was generated by irradiating the left eyes of mice with UVB light(360μW/cm2),and the lens capsules of mice were collected.Transfection efficiencies were verified by real-time qPCR,and the expression levels of miR-125b and mRNA of its predictive target gene BAK1 were detected.The expression of BAK1 protein was detected by Western blotting analysis,and the apoptosis rate of human lens epithelial cells was detected by TUNEL apoptosis assay.Results The results indicated that when compared with normal lens epithelial cells,the expression level of miR-125b in the apoptosis model of human lens epithelial cells was significantly decreased,along with observed increases in the expression of BAK1 protein and the apoptosis rate in human lens epithelial cells.Also,in the cells transfected with miR-125b mimics,the expression levels of miR-125b was significantly increased,the expression of BAK1 protein was decreased,and the apoptosis rate was decreased when compared to cells transfected with control RNA.In cells transfected with miR-125b inhibitor,the expression of miR-125b was significantly decreased,expression of BAK1 protein was significantly increased,and apoptosis was increased,when compared to the cells transfected with miR-125b inhibitor controls.Finally,a comparison between non-irradiated and UVB-irradiated mice models were made and the expression of miR-125b in the lens capsules was significantly decreased,expression of BAK1 protein was increased,and apoptosis was increased in the UVB-irradiated mice model group.Conclusion miR-125b affects the apoptosis of human lens epithelial cells through targeted regulation of BAK1,thus regulating the pathogenesis of cataract,and this could become a new strategy for non-operative targeted treatment of cataracts.