LncRNA GAS5靶向miR-103减轻3T3L1脂肪细胞胰岛素抵抗的作用机制 #br#

目的 探究长链非编码RNA（LncRNA）生长抑制特异因子5（GAS5）靶向微小RNA（miR）-103，从而减轻3T3L1脂肪细胞胰岛素抵抗（IR）的机制。方法 培养并诱导分化3T3L1小鼠前脂肪细胞，油红O染色鉴定细胞分化情况。建立3T3L1脂肪细胞IR模型。实验分为对照组、模型组、空载体组（转染pEGFP-C1空载体）、GAS5过表达组（转染 pEGFP-C1-GAS5 载体）、GAS5 过表达+mimic NC 组（转染 pEGFP-C1-GAS5+mimic NC）、GAS5 过表达+miR-103 mimic组（转染pEGFP-C1-GAS5+miR-103 mimic）。实时荧光定量PCR检测细胞中GAS5、miR-103 mRNA水平；液体闪烁法检测葡萄糖摄取能力；蛋白质免疫印迹检测细胞中胰岛素受体底物-1（IRS-1）、p-IRS-1、过氧化物酶体增殖物激活受体γ（PPARγ）、葡萄糖转运蛋白4（GLUT4）、蛋白激酶B（AKT）、p-AKT蛋白表达水平；双荧光素酶鉴定miR-103与GAS5的靶向位点。结果 诱导后脂肪细胞呈圆形、胞体变大，胞浆丰富，含有大量脂滴，油红染色明显，呈“指环样”结构，模型构建成功。与对照组比较，模型组、空载体组、GAS5过表达+miR-103 mimic 组细胞中GAS5mRNA水平、葡萄糖摄取能力、p-IRS-1/IRS-1、PPARγ、GLUT4、p-AKT/AKT蛋白水平降低，细胞中miR-103 mRNA水平升高（P＜0.05）；与模型组、空载体组比较，GAS5过表达组、GAS5过表达+mimic NC组细胞中GAS5 mRNA水平、葡萄糖摄取能力、p-IRS-1/IRS-1、PPARγ、GLUT4、p-AKT/AKT蛋白水平升高，而miR-103 mRNA水平降低（P＜0.05）；与GAS5过表达组、GAS5过表达+mimic NC组比较，GAS5过表达+miR-103 mimic组细胞中GAS5 mRNA水平、葡萄糖摄取能力、p-IRS-1/IRS-1、PPARγ、GLUT4、p-AKT/AKT 蛋白水平降低，miR-103 mRNA 水平升高（P＜0.05）。miR-103与GAS5存在互补的结合位点并经双荧光素酶靶向关系验证。结论 过表达GAS5后靶向下调miR-103的表达可减轻3T3L1脂肪细胞IR。

Mechanism of LncRNA GAS5 targeting miR-103 to reduce insulin resistance in3T3L1 adipocytes #br#

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 mousepreadipocytes 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 vectorgroup (transfected with pEGFP-C1 empty vector), the GAS5 over-expression group (transfected with pEGFP-C1-GAS5vector), the GAS5 over-expression + mimic NC group (transfected with pEGFP-C1-GAS5+mimic NC) and the GAS5overexpression+miR-103 mimic group (transfected with pEGFP-C1-GAS5+miR-103 mimic). Real-time fluorescentquantitative PCR was used to detect the levels of GAS5 mRNA and miR-103 in cells. liquid scintillation method was used todetect 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 kinaseB (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 lipiddroplets. 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-103mimic group (P＜0.05), the miR-103 mRNA level in cells increased (P＜0.05). Compared with the model group and theempty vector group, the GAS5 mRNA level, glucose uptake capacity, p-IRS-1/IRS-1, PPARγ, GLUT4 and p-AKT/AKTprotein 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 theGAS5 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), themiR-103 mRNA level increased (P＜0.05). The complementary binding sites of MiR-103 and GAS5 were verified by thedual luciferase targeting relationship. Conclusion Targeted down-regulation of miR-103 expression after over-expressionof GAS5 can reduce IR of 3T3L1 adipocytes.