miRNA-126调控自噬减轻高糖诱导血管内皮细胞损伤的作用机制  

Th e mechanism of miRNA-126 regulating autophagy to alleviate high glucose induced damage to vascular endothelial cells

作　　者：郑伟英[1] 刘心雨 刘俊汝 李萌 包慧兰[1] 楼时先[1] 金启辉[2] Zheng Weiying

机构地区：[1]浙江省金华市人民医院,321000 [2]浙江大学医学院附属第二医院,310009

出　　处：《浙江临床医学》2023年第7期971-974,977,共5页Zhejiang Clinical Medical Journal

基　　金：金华市科技计划项目(2019-4-035)。

摘　　要：目的探讨miRNA-126对高糖诱导的内皮细胞自噬与凋亡的影响及分子机制。方法采用高糖(HG)诱导HUVECs模型,随机分为4组,即正常葡萄糖(5.5 mmo/L)对照组,HG模型组,miRNA-126-5p mimics组(转染miRNA-126-5p mmics组),miRNA-126-5p inhibitor组(转染miRNA-126 inhibitor组)。HG模型组即终浓度为33.3 mmol/L的葡萄糖诱导24 h,miRNA-126-5p mimics组是miRNA-126-5p mimics转染至HUVECs细胞,48 h后在培养基中加入33.3 mmol/L(终浓度)葡萄糖,培养24 h。miRNA-126-5p inhibitor组是miRNA-126-5p inhibitor转染至HUVECs细胞,48 h后在培养基中加入33.3 mmol/L(终浓度)的葡萄糖,培养24 h。再利用miRNA-126-5p mimics组模型,分别予自噬抑制剂3-甲基腺嘌呤(3-methyl adenine,3-MA)5 mmol/L、ERK通路抑制剂(U-0126)10 mmol/L干预24 h。RT-qPCR检测细胞中miRNA-126-5p的表达水平,CCK-8检测细胞活性,划痕实验检测细胞迁移能力,Westernblot检测细胞培养液中血管内皮生长因子(VEGF)和肝细胞生长因子(HGF),p-ERK、ERK蛋白,p-AKT、AKT蛋白,Bax、Bcl-2蛋白,cleaved-caspase-3蛋白,Beclin-1和Lc3蛋白含量。结果与正常组比较,HG模型细胞中miRNA-126水平,p-ERK/ERK蛋白水平低于正常对照组(P<0.05);与HG模型组相比较,miRNA-126-5p mimics组细胞中miRNA-126、p-ERK/ERK蛋白表达明显升高(P<0.01),细胞中Bax/Bcl-2蛋白,cleaved caspase-3蛋白表达下降(P<0.05),Beclin-1和Lc3蛋白的表达水平升高(P<0.01),细胞活性增强(P<0.01),细胞迁移率提高(P<0.01),VEGF水平升高(P<0.05);与miRNA-126-5p inhibitor组比较,miRNA-126-5p mimics组细胞中miRNA-126、pERK/ERK蛋白表达升高(P<0.01),细胞中Bax/Bcl-2蛋白,cleaved caspase-3蛋白表达下降(P<0.05),Beclin-1和Lc3蛋白表达水平升高(P<0.01),细胞活性增强(P<0.05),细胞迁移率提高(P<0.05),VEGF水平升高(P<0.01);在miRNA-126-5p mimics组中加入3-MA后凋亡蛋白表达明显下降(P<0.01),加入ERK抑制剂后,自噬蛋白表达明显下降(P<0.01)。结论miRNA-126可通过激活ERK信号通Objective To explore the effect and molecular mechanism of miRNA-126 on autophagy and apoptosis of endothelial cells induced by high glucose(HG).Methods Human umbilical vein endothelial cell(HUVEC)models induced by HG were randomly divided into four groups:the control group(normal glucose 5.5 mmol/L),the HG model group,the miRNA-126-5p mimic group,and the miRNA-126-5p inhibitor group.The HG model group was induced by glucose at a final concentration of 33.3 mmol/L for 24 h.In the miRNA-126-5p mimic group,the miRNA-126-5p mimic was transfected into HUVECs,and glucose was added(final concentration 33.3 mmol/L)into the medium after 48 h and cultured for 24 h.In the miRNA-126-5p inhibitor group,the miRNA-126-5p inhibitor was transfected into HUVECs.After 48 h,glucose(final concentration 33.3 mmol/L)was added to the medium and cultured for 24 h.Using the miRNA-126-5p mimic group model,the autophagy inhibitor(3-methyladenine,3-MA)5 mmol/L and extracellular regulated protein kinases(ERK)pathway inhibitor(U-0126)10 mmol/L interfered for 24 h.The expression of miRNA-126-5p in cells,cell activity,and cell migration ability were detected by reverse transcriptase/quantitative polymerase chain reaction(RT/qPCR),cell counting kit-8(CCK-8),and scratch test,respectively.Western blot was used to detect the content of vascular endothelial growth factor(VEGF),hepatocyte growth factor(HGF),p-ERK,ERK protein,p-AKT,AKT protein,Bax,Bcl-2 protein,cleaved caspase-3 protein,Beclin-1 and light chain 3(LC3)protein in cell culture medium.Results Compared with the control group,the expression of miRNA-126 and p-ERK/ERK protein was significantly lower in the HG model group(P<0.01).Compared with the HG model group and the miRNA-126-5p inhibitor group,the expression of miRNA-126 and p-ERK/ERK protein significantly increased in the miRNA-126-5p mimic group(P<0.01),the expression of Bax/Bcl-2 protein and cleaved caspase-3 protein in cells decreased(P<0.01),and the expression of Beclin-1 and LC3 protein,cell activity,cell migration rate,and VEGF al

关 键 词：高糖 内皮细胞 miRNA-126 细胞外调节蛋白激酶 自噬 凋亡 

分 类 号：R58[医药卫生—内分泌]