Effect of long-chain non-coding RNA GAS5 on insulin secretion of islet cells by targeting miR-29, miR-96, and miR-208
10.3760/cma.j.cn311282-20200914-00634
- VernacularTitle: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
- Author:
Yijia LI
1
;
Hui CHEN
Author Information
1. 中南大学湘雅三医院检验科，长沙 410007
- Keywords:
Diabetes mellitus, type 2;
Long non-coding RNA;
GAS5;
Insulin secretion;
Gene therapy
- From:
Chinese Journal of Endocrinology and Metabolism
2021;37(8):745-751
- CountryChina
- Language:Chinese
-
Abstract:
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 pancreatic β-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 islet β-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 interaction was validated with dual luciferase reporter gene assay. shGAS5 significantly decreased the expressions of INSR, IRS-1 and PI3K( P were 0.022, 0.038, and 0.009), while overexpressed GAS5 significantly upregulated the expressions of INSR, IRS-1 and PI3K at both mRNA and protein levels( P were 0.024, 0.045, and 0.016). Conclusion:GAS5 could stimulate insulin secretion of islet cell through its inhibitry regulationor of expressions of miR-29, miR-96, and miR-208, therely up-regulating INSR, IRS-1, and PI3K that may be the potential targets of these miRNAs, and stimulate insulin secretion of islet cells.
