微小RNA⁃199⁃3p通过一磷酸腺苷活化蛋白激酶/雷帕霉素靶蛋白通路抑制高糖诱导的白色脂肪细胞分化  

MicroRNA-199-3p inhibits high glucose-induced differentiation of white adipocytes via the adenosine 5’-monophosphate-activated protein kinase/mammalian target-of-rapamycin pathway

作　　者：胡淑芳[1] 刘玄林 杨丽[1] Hu Shufang;Liu Xuanlin;Yang Li(Department of Endocrinology,Hankou Hospital of Wuhan City,Wuhan 430012,China)

机构地区：[1]湖北省武汉市汉口医院内分泌科,430012

出　　处：《临床内科杂志》2021年第1期51-55,共5页Journal of Clinical Internal Medicine

基　　金：武汉市卫生和计划生育委员会科研项目(WX15D39、WZ16C22)。

摘　　要：目的探讨微小RNA(miR)-199a-3p与2型糖尿病(T2DM)病理特征关系及其对白色脂肪细胞分化和脂肪炎症调控的分子机制。方法收集2015年6月~2018年4月就诊于我院的131例T2DM患者(T2DM组)与146例健康志愿者(NC组)的临床资料。采用RT-qPCR检测外周血has-miR-199a-5p、has-miR-199a-3p、has-miR-199b-5p和has-miR-199b-3p水平;采用ELISA检测肿瘤坏死因子-α(TNF-α)、单核细胞趋化蛋白-1(MCP-1)、瘦素、脂联素、内脂素、抵抗素和白细胞介素-6(IL-6)含量,并探讨其与miR-199a-3p的相关性。3T3-L1细胞经高糖、低糖和分化诱导处理后,采用RT-qPCR检测3T3-L1细胞中has-miR-199a-3p含量,ELISA检测TNF-α和IL-6含量;油红O染色观察miR-199a-3p类似物(mimics)处理高糖诱导的3T3-L1脂滴形成;Western blot检测mimics干预高糖处理3T3-L1细胞中胰岛素受体底物-1(IRS-1)、磷酸化磷脂酰肌醇-3激酶P85亚基(p-P85)、磷酸化蛋白激酶B(p-AKT)、磷酸化糖原合成酶激酶3β(p-GSK3b)、葡萄糖转运体-4(GLUT-4)、磷酸化信号传导与转录激活因子-3(p-STAT3)、磷酸化一磷酸腺苷活化蛋白激酶(p-AMPK)、磷酸化雷帕霉素靶蛋白(p-mTOR)、NAD-依赖性去乙酰化酶(SIRT1)和过氧化物酶体增殖物激活受体-γ(PPAR-γ)蛋白表达。采用Pearson相关分析评估各变量间的相关性。结果T2DM组患者血浆miR-199a-3p、miR-199b-5p和miR-199b-3p均明显低于NC组(P<0.05)。重度T2DM组患者miR-199a-3p含量明显低于轻、中度T2DM组(P<0.05)。T2DM组血浆miR-199a-3p含量与空腹胰岛素(FIns)、空腹血糖(FPG)、胰岛素抵抗指数(HOMA-IR)、TNF-α及IL-6呈负相关(P<0.05)。高糖组miR-199a-3p含量高于对照组,分化的脂肪细胞内miR-199a-3p含量明显高于3T3-L1细胞(P<0.05),mimics组油脂滴数量明显低于阴性对照组。高糖+抗miR-199a-3p组的p-AMPK、p-mTOR、SIRT-1和PPAR-γ蛋白表达均明显低于高糖组(P<0.05),两组IRS-1、p-P85、p-AKT、p-GSK3b和Glut-4蛋白表达比较差异均无统计学Objective To investigate the relationship between microRNA(miR)-199a-3p and type 2 diabetes mellitus(T2DM)and its effect on white adipocyte differentiation and inflammation.Methods The clinical and pathological data of 131 T2DM patients and 146 healthy volunteers(NC group)from June,2015 to April,2018 were collected.RT-qPCR was used to detect the levels of has-miR-199a-5p,has-miR-199a-3p,has-miR-199b-5p and has-miR-199b-3p in peripheral blood.Tumor necrosis factor-α(TNF-α),monocyte chemoattractant protein-1(MCP-1),leptin,adiponectin,visfatin,resistin and interleukin-6(IL-6)contents were measured by ELISA,and the correlation with miR-199a-3p was detected.After treated with high glucose,low glucose and differentiation induction,the content of has-miR-199a-3p in 3T3-L1 cells was detected by RT-qPCR.TNF-αand IL-6 levels were detected by ELISA,as well as differentiation induction medium treatment.Oil red O staining was used to detect lipid droplets in 3T3-L1 cells after high-glucose and miR-199a-3p analogues(mimics)treatment.Western blotting was used to detect insulin receptor substrate-1(IRS-1),phosphorylation Phosphatidylinositol-3 kinase P85 subunit(p-P85),phosphorylated protein kinase B(p-AKT),phosphorylated glycogen synthase kinase 3β(p-GSK3β),glucose transporter-4(GLUT-4),phosphorylated signal transduction and transcription activator-3(p-STAT3),phosphorylated adenosine monophosphate activated protein kinase(p-AMPK),phosphorylated target of rapamycin(p-mTOR),NAD-dependent acetylase(SIRT1)and peroxisome proliferator activated receptor-γ(PPAR-γ)protein expression after miR-199a-3p mimics treatment in high glucose treated 3T3-L1 cells.Pearson analysis was used to evaluate the correlation.Results Plasma miR-199a-3p,miR-199b-5p and miR-199b-3p in the T2DM group were significantly lower than those of NC group(P<0.05).The content of miR-199a-3p was significantly lower in severe T2DM group compared with mild to moderate T2DM group(P<0.05).Plasma miR-199a-3p content in T2DM group was negatively correlated with fasti

关 键 词：2型糖尿病 微小RNA-199a-3p 脂肪再生 脂肪因子 炎症因子 一磷酸腺苷活化蛋白激酶/雷帕霉素靶蛋白信号通路 

分 类 号：R589.2[医药卫生—内分泌]