抑制miR-34a减少高糖代谢记忆所致视网膜色素上皮细胞SIRT1下调和凋亡  

Inhibition of miR-34a allviates downregulation of SIRT1 and apoptosis by high glucose metabolic memory in retinal pigment epithelial cells

作　　者：黄焱[1] 郑卫东[2] 尤加慧 徐国兴[2] 

机构地区：[1]福建医科大学医技学院眼视光学系 [2]福建医科大学附属第一医院眼科,福州350004

出　　处：《中国组织化学与细胞化学杂志》2017年第5期462-467,共6页Chinese Journal of Histochemistry and Cytochemistry

基　　金：福建省自然科学基金(2016J01155);福建医科大学教授基金(JS14013)

摘　　要：目的探讨mi R-34a在视网膜色素上皮(retinal pigment epithelium,RPE)细胞高糖"代谢记忆"中的作用及其机制,为糖尿病视网膜病的防治提供新的策略。方法 RPE细胞用高糖培养4d后换用正常糖浓度培养4d,模拟糖尿病高糖代谢记忆模型,用mi R-34a mimic和mi R-34a inhibitor改变高糖代谢记忆RPE细胞内mi R-34a表达,实时定量PCR检测mi R-34a表达水平,免疫细胞化学和Western blot检测组蛋白去乙酰化酶Sirtuin 1(SIRT1)水平,annexin V-PI法流式细胞术检测细胞凋亡。结果实时定量PCR检测显示,高糖培养和代谢记忆可显著升高RPE细胞内mi R-34a表达水平,mi R-34a mimic可进一步显著上调代谢记忆RPE细胞内mi R-34a表达水平,mi R-34a inhibitor可显著下调代谢记忆RPE细胞内mi R-34a水平;免疫细胞化学染色显示,高糖培养和代谢记忆可使RPE细胞内STRT1免疫反应性显著降低,过表达mi R-34a进一步下调代谢记忆RPE细胞内STRT1免疫反应性,而mi R-34a inhibitor则可抑制代谢记忆对RPE细胞内STRT1免疫反应性的下调作用;流式细胞术检测发现,高糖培养和代谢记忆使RPE细胞凋亡明显增加,过表达mi R-34a进一步增加代谢记忆RPE细胞凋亡,而mi R-34a inhibitor则显著抑制代谢记忆RPE细胞凋亡的增加。结论 mi R-34a抑制剂能通过抑制高糖代谢记忆对RPE细胞SIRT1水平的下调和细胞凋亡的增加,部分逆转高糖代谢记忆效应,防治糖尿病视网膜病变。Objective To investigate the role of miR-34a in retinal pigment epithelium （RPE） cells with high glucose metabolic memory and explore new strategy for the prevention and treatment of diabetic retinopathy. Methods RPE cells were cultured under various conditions. The high glucose metabolic memory model of diabetes mellitus was simulated through culturing cells in high glucose for 4 days followed by 4 days in normal glucose medium. The expression of miR-34a was measured by quantitative real-time PCR （qRT-PCR）. The level of histone deacetylase Sirtuin 1 （SIRT1） was evaluated using immunocytochemistry and Western blot. Apoptotic cells were detected by flow cytometry using Annexin V/Propidium iodide double staining. Results qRT-PCR showed that miR-34a expression in RPE cells was significantly increased in high glucose and metabolic memory groups. Its expression in RPE cells with metabolic memory could be further modulated, e.g. up-regulated by MiR-34a mimic or down-regulated by inhibitor. Immunocytochemistry staining showed that STRT1 expression in RPE cells was significantly decreased in high glucose culture and metabolic memory groups. Overexpressing miR-34a further down-regulated STRT1 in metabolic memory RPE cells, while miR-34a inhibitor partially abolished the inhibitory effects mediated by metabolic memory. Flow cytometry showed that apoptotic RPE cells significantly increased in high-glucose culture and metabolic memory groups, which could be further increased by miR-34a overexpression but reduced by miR-34a inhibitor in RPE cells with metabolic memory. Conclusion MiR-34a inhibitor can partially reverse high glucose metabolic memory effect in RPE cells and prevent diabetic retinopathy likely through reducing SIRT1 expression and apoptosis.

关 键 词：高糖 代谢记忆 视网膜色素上皮细胞 MIR-34A Sirtuin1 

分 类 号：R774.1[医药卫生—眼科]