miRNA-146a在视网膜色素上皮细胞老化及老年性黄斑变性中的功能研究

目的 检测miRNA-146a(miR-146a)在视网膜色素上皮细胞(retinal pigment epithelial cells,RPE)衰老及老年性黄斑变性(age-related macular degeneration,AMD)中的表达水平,并探讨其通过抑制VEGF-A表达,参与调控AMD病变进程的机制.方法 qRT-PCR检测年龄为2个月、8个月、12个月、18个月和24个月小鼠的RPE及75岁晚期“湿”性AMD患者眼球中miR-146以及VEGF-A的mRNA表达水平.Western Blotting检测VEGF-A蛋白表达水平.在ARPE-19细胞系中转染miR-146a,通过qRT-PCR及Western Blotting检测miR-146a对VEGF-A mRNA及蛋白水平的影响.结果 在自然衰老的RPE中,miR-146的表达呈现随年龄增加逐渐上升的趋势,与2个月的小鼠相比,在18个月及24个月的小鼠中miR-146a水平上升约8倍及24倍.而VEGF-A的表达呈现下降趋势,与对照相比,24个月的小鼠RPE中的VEGF-A相对水平由2个月时的1.5倍降为0.8倍.但是在AMD患者中,病灶部位的miR-146a较病灶旁侧表达下降14.5倍,VEGF-A表达上升.在ARPE-19细胞中过表达miR-146a抑制了VEGF-A的mRNA及蛋白表达水平.结论 本实验结果将miR-146a与RPE衰老及AMD的发生联系在一起,未来有可能成为RPE衰老及AMD早期诊断的新的分子标记物,为制定临床治疗策略提供参考.

Function of miR-146a in retinal pigment epithelial cells aging and age-related macular degeneration

Objective To investigate the expression level of miRNA-146a (miR-146a) in retinal pigment epithelial (RPE) cells aging and age-related macular degeneration (AMD),and discuss its regulation mechanism of AMD by repressing VEGF-A.Methods The expressions of miR-146 and VEGF-A were examined by qRT-PCR in RPE cells in mice aged 2 months,8 months,12 months,18 months or 24 months,and in RPE cells from 75 years old AMD patients.The protein level of VEGF-A was also detected by Western Blotting.Finally,the effects of overexpression of miR-146a in APRE-19 cell line on expression of VEGF-A was checked.Results MiR-146 was up-regulated to 8 times or 24 times at 18 months or 24 months aged mice,and the expression of VEGFA was down-regulated in aging RPE from 1.5 times to 0.8 times.However,the expression of miR-146 decreased to 14.5 times and VEGF-A increased in RPE cells of AMD.In cultured cells,overexpression of miR-146a inhibited the expression of VEGF-A.Conclusion Up-regulation of miR-146a in aging RPE cells and its down-regulation in AMD suggest a potential of miR-146a as molecular marker.MiR-146a overexpression inhibits the expression of VEGF-A,supporting a potential clinical treatment of miR-146 in AMD.