miR-7a-5p负调控Rac1表达参与HepG2细胞胰岛素抵抗

miR-7a-5p contributes to insulin resistance in HepG2 cells through nega⁃tively regulating Rac1

目的:探讨软脂酸诱导胰岛素抵抗的HepG2细胞中微小RNA-7a-5p(microRNA-7a-5p,miR-7a-5p)是否通过调控Ras相关C3肉毒毒素底物1(Ras-related C3 botulinum toxin substrate 1,Rac1)表达参与细胞胰岛素抵抗。方法:应用生物信息学分析预测其下游靶分子,构建含Rac13'端非翻译区的野生型及突变型hsa-Rac1萤光素酶报告质粒,与miR-7a-5p表达慢病毒或对照空载慢病毒共转染293T细胞,检测萤光素酶活性。HepG2细胞分为软脂酸诱导的胰岛素抵抗组和正常对照组,RT-qPCR分析两组细胞miR-RNA-7a-5p的表达,Western blot分析下游靶分子蛋白的表达。HepG2细胞分为干扰慢病毒转染组和对照慢病毒转染组,转染之后,软脂酸诱导两组细胞胰岛素抵抗,分析两组HepG2细胞中脂质堆积及葡萄糖消耗情况。结果:生物信息学预测Rac1为miR-7a-5p下游分子。野生型hsa-Rac1萤光素酶报告质粒与miR-7a-5p表达慢病毒共转染后,萤光素酶较对照组显著降低(P<0.05)。胰岛素抵抗的HepG2与对照细胞组相比,miR-7a-5p表达显著下调(P<0.05),Rac1蛋白表达显著上调(P<0.05)。较对照相比,Rac1 mRNA的表达抑制增加了胰岛素抵抗HepG2细胞的葡萄糖消耗(P<0.05),降低了胞质内脂质堆积(P<0.05)。结论:miR-7a-5p的一个潜在靶点是Rac1。miR-7a-5p通过负调节Rac1表达参与软脂酸诱导的HepG2细胞胰岛素抵抗。

AIM:To investigate the effect of microRNA-7a-5p(miR-7a-5p)on insulin resistance induced by palmitic acid in HepG2 cells.METHODS:Downstream targeting molecules of miR-7a-5p were predicted by bioinformatics.Luciferase reporter plasmids of wild-type and mutant hsa-Rac1(Ras-related C3 botulinum toxin substrate 1)containing 3'-untranslated region were constructed and co-transfected 293T cells with miR-7a-5p lentivirus or its control mimic,and then the luciferase activity was detected.The HepG2 cells were randomly divided into palmitic acid-induced insulin resistance group and normal control group.The miR-7a-5p expression was analyzed by RT-qPCR,and the expression of downstream targeting molecule was detected by Western blot the 2 groups.The HepG2 cells were randomly divided into interference lentivirus group and control mimic group.After transfection,the cells were treated with palmitic acid to induce insulin resistance.Glucose consumption and fatty acid accumulation were measured in the 2 groups.RESULTS:The Rac1 mRNA was forecasted to be direct targeting molecule of miR-7a-5p.Wild-type hsa-Rac1 luciferase reporter plasmids co-transfected with miR-7a-5p lentivirus significantly decreased the luciferase activity when compared with control group(P<0.05).The miR-7a-5p expression was significantly down-regulated(P<0.05),and Rac1 protein expression significantly was up-regulated(P<0.05)in HepG2 cells with insulin resistance compared with control group.Compared with control group,the suppression of Rac1 mRNA expression by hsa-Rac1 interference lentivirus increased glucose con‐sumption and decreased fatty acid accumulation in HepG2 cells with insulin resistance.CONCLUSION:The potential target molecule of miR-7a-5p is Rac1 mRNA.miR-7a-5p contributes to palmitic acid-induced insulin resistance in HepG2 cells through negatively regulating Rac1.