关键词: 糖尿病, 2型, RNA, 长链非编码, 微RNAs, 3T3-L1细胞, 胰岛素抵抗, 生长抑制特异因子5, 微小RNA-103

Abstract: Objective To explore the mechanism of long non-coding RNA (LncRNA) growth arrest-specific 5 (GAS5) targeting microRNA (miR) -103 to reduce 3T3L1 adipocyte insulin resistance (IR). Methods The 3T3L1 mouse preadipocytes were cultured and induced to differentiation, and oil red O staining was used to identify cell differentiation. The 3T3L1 adipocyte IR model was established. Cells were divided into the control group, the model group, the empty vector group (transfected with pEGFP-C1 empty vector), the GAS5 over-expression group (transfected with pEGFP-C1-GAS5 vector), the GAS5 over-expression + mimic NC group (transfected with pEGFP-C1-GAS5+mimic NC) and the GAS5 overexpression+miR-103 mimic group (transfected with pEGFP-C1-GAS5+miR-103 mimic). Real-time fluorescent quantitative PCR was used to detect the levels of GAS5 mRNA and miR-103 in cells. liquid scintillation method was used to detect glucose uptake capacity. Western blot assay was used to detect the expression levels of insulin receptor substrate-1 (IRS-1), p-IRS-1, peroxisome proliferator-activated receptor γ (PPARγ), glucose transporter 4 (GLUT4), and protein kinase B (AKT) and p-AKT proteins. Dual luciferase was used to identify the targeting sites of miR-103 and GAS5. Results After induction, the cells were round, the cell body enlarged, the cytoplasm was rich and contained a large number of lipid droplets. The oil red staining was obvious, showing‘finger-ring-like’structure. The model was successfully constructed. Compared with the control group, the GAS5 mRNA level, glucose uptake capacity, p-IRS-1/IRS-1, PPARγ, GLUT4 and pAKT/AKT protein levels decreased in the model group, the empty vector group and the GAS5 over-expression+miR-103 mimic group (P＜0.05), the miR-103 mRNA level in cells increased (P＜0.05). Compared with the model group and the empty vector group, the GAS5 mRNA level, glucose uptake capacity, p-IRS-1/IRS-1, PPARγ, GLUT4 and p-AKT/AKT protein levels increased in the GAS5 over-expression group and the GAS5 over-expression+mimic NC group (P＜0.05), while the miR-103 mRNA level in cells decreased (P＜0.05). Compared with the GAS5 over-expression group and the GAS5 over-expression + mimic NC group, the GAS5 mRNA level, glucose uptake capacity, p-IRS-1/IRS-1, PPARγ, GLUT4 and p-AKT/AKT protein levels decreased in the GAS5 over-expression + miR-103 mimic group (P＜0.05), the miR-103 mRNA level increased (P＜0.05). The complementary binding sites of MiR-103 and GAS5 were verified by the dual luciferase targeting relationship. Conclusion Targeted down-regulation of miR-103 expression after over-expression of GAS5 can reduce IR of 3T3L1 adipocytes.

Key words: diabetes mellitus, type 2, RNA, long noncoding, microRNAs, 3T3-L1 cells, insulin resistance, growth arrest-specific 5, microRNA-103