长链非编码RNA GAS5靶向调控miR-29、miR-96和miR-208影响胰岛β细胞分泌胰岛素功能的机制研究  

Effect of long-chain non-coding RNA GAS5 on insulin secretion of islet cells by targeting miR-29,miR-96,and miR-208

作　　者：李颖佳[1] 陈辉[1] Li Yijia;Chen Hui(Department of Emergency,The Third Xiangya Hospital,Central South University,Changsha 410007,China)

机构地区：[1]中南大学湘雅三医院检验科,长沙410007

出　　处：《中华内分泌代谢杂志》2021年第8期745-751,共7页Chinese Journal of Endocrinology and Metabolism

基　　金：湖南省自然科学基金(S2019JJQNJJ2291)。

摘　　要：目的:研究长链非编码RNA GAS5(long non-coding RNA GAS5,GAS5)靶向调控miR-29、miR-96和miR-208促进胰岛β细胞分泌胰岛素的作用及其机制。方法:采用Q-PCR法检测122名健康人血清和88例2型糖尿病(T2DM)患者血清以及大鼠胰岛细胞瘤株INS-1832/13中,长链非编码RNA GAS5与miR-29、miR-96和miR-208的表达水平及其相关性。通过慢病毒载体构建,沉默和过表达GAS5观察对胰岛β细胞分泌胰岛素功能的影响。采用生物信息学预测GAS5与miR-29、miR-96和miR-208有互补结合位点,用荧光素酶报告系统验证其靶向关系。在胰岛β细胞中共转染GAS5 shRNA,用上述方法检测其对胰岛素受体(insulin receptor,INSR)、胰岛素受体底物1(insulin receptor substrate-1,IRS-1)和磷酸肌醇3-激酶(phosphatidylinositide 3-kinase,PI3K)水平的影响,从而揭示GAS5刺激胰岛β细胞分泌胰岛素的作用机制。结果:与健康人血清相比,2型糖尿病患者血清中GAS5的表达低于健康对照组(t=4.632,P<0.01),miR-29、miR-96和miR-208高于健康对照组(t分别为7.832、9.164、12.359,均P<0.01),而且GAS5水平与miR-29、miR-96和miR-208呈负相关(r分别为-0.50、-0.47、-0.70)。慢病毒过表达GAS5导致葡萄糖刺激的胰岛素分泌增加,胰岛素含量增加。相反,GAS5的下调导致葡萄糖刺激的胰岛素分泌和胰岛素含量显著降低。利用生物信息学工具,筛选了miR-29、miR-96和miR-208作为GAS5的靶点,并通过双荧光素酶报告基因实验验证GAS5可以通过靶向抑制miR-96、miR-29和miR-208的表达发挥作用。shGAS5显著降低INSR、IRS-1和PI3K的表达(P分别为0.022、0.038、0.009),而过表达GAS5在mRNA和蛋白水平上均显著上调INSR、IRS-1和PI3K的表达(P分别为0.024、0.045、0.016)。结论:GAS5可能通过靶向调控miR-29、miR-96和miR-208的表达,进而对这些miRNA可能存在的负调控因子INSR、IRS-1和PI3K等的表达进行正向调控,刺激胰岛β细胞分泌胰岛素。Objective To investigate the role and mechanism of long non-coding RNA GAS5 in the targeted regulation of miR-29,miR-96,and miR-208 in promoting insulin secretion of pancreaticβ-cells.Methods Q-PCR was used to detect the expression of miR-29,miR-96,and miR-208 in sera of 122 healthy subjects and 88 type 2 diabetic patients;and so of long non-coding RNA GAS5 and miR-208 in the rat islet cell tumor strain ins-1832/13.Effects of silencing and overexpressing GAS5 on insulin secretion of isletβ-cells by lentiviral vector construction were observed.Bioinformatics was used to predict that GAS5 had complementary binding sites with miR-29,miR-96,and miR-208,which was further verified by luciferase reporting system.GAS5 siRNA was co-transfected with miR-29,miR-96,and miR-208 inhibitors,and the effect of GAS5 on insulin receptor(INSR),insulin receptor substrate(irs-1)and PI3K levels was detected by the above method,so as to reveal the effect of GAS5 on insulin secretion in islet cells.Results The expression of GAS5 in serum of T2DM patients was lower than that of healthy control group(t=4.632,P<0.01),and expression of miR-29,miR-96,and miR-208 were higher than those of healthy control group(t were 7.832,9.164,and 12.359,all P<0.01).GAS5 level was negatively correlated with miR-29,miR-96,and miR-208(r were-0.50,-0.47,and-0.70,respectively).GAS5 expression was significantly decreased in serum of type 2 diabetic patients compared with that of in healthy subjects.Overexpression of GAS5 by lentivirus resulted in increased glucose-stimulated insulin secretion and increased insulin concentration compared to negative control.In contrast,knockdown of GAS5 led to significant reduction of glucose-stimulated insulin secretion and insulin concentration.GAS5 levels were negatively correlated with miR-29,miR-96,and miR-208 in serum samples of type-2 diabetes patients.GAS5 can negatively regulate the expression of miR-96,miR-29,and miR-208.By bioinformatics tools,we screened miR-29,miR-96 and miR-208 as targets of GAS5,and their interact

关 键 词：糖尿病 2型 长链非编码RNA GAS5 胰岛素分泌 基因治疗 

分 类 号：R587.1[医药卫生—内分泌]