miR-132-3p靶向UCP2调节绵羊前体脂肪细胞分化的研究

摘要

旨在研究miR-132-3p对绵羊前体脂肪细胞中UCP2基因的表达调控机制.本研究以绵羊皮下脂肪细胞为研究对象,检测miR-132-3p与UCP2在绵羊前体脂肪细胞中的作用机制;利用生物信息学软件预测miR-132-3p与UCP2的结合位点,并通过双荧光素酶报告试验验证miR-132-3p与UCP2的靶标关系;在绵羊前体脂肪细胞中过表达miR-132-3p,用RT-qPCR、Western blotting技术检测过表达后UCP2 mRNA和蛋白的表达水平;最后,采用RT-qPCR检测绵羊前体脂肪细胞分化过程中UCP2和miR-132 3p的表达.结果表明,miR-132-3p在UCP2 3'-UTR上存在结合位点,并显著下调UCP2 3'-UTR重组双荧光质粒的相对荧光活性(P＜0.05);过表达miR-132-3p显著下调UCP2 mRNA的表达(P＜0.05),且明显降低UCP2蛋白的表达量;绵羊前体脂肪细胞分化中期miR-132-3p与UCP2的表达存在负相关关系.综上表明,miR-132-3p通过特异性结合UCP2 3'-UTR抑制UCP2在mRNA和蛋白水平的表达,在一定程度上促进绵羊前体脂肪细胞的分化.%This study aimed to explore the regulatory mechanism of miR-132-3p targeting UCP2 expression in ovine preadipocytes.In this study,ovine subcutaneous fat cells were used to investigate the mechanism of miR-132-3p targeting UCP2 in ovine preadipocytes.The binding sites of miR-132-3p to UCP2 were predicted by bioinformatics softwares,and were verified via double luciferase report system.Expression of UCP2 mRNA and protein were detected by RT-qPCR and Western blotting,respectively after overexpressing miR-132-3p.The expression of UCP2 mRNA and miR-132-3p were detected by RT-qPCR during differentiation stage in ovine preadipocyte.The results showed that miR-132-3p had binding site to UCP2 3'-UTR and significantly downregulated the relative fluorescence activity of recombinant double fluorescent plasmid(P＜0.05).Overexpressing miR-132-3p significantly down-regulated both UCP2 mRNA(P＜0.05) and protein expressions.At last,we found a negative correlation between the expression of miR-132-3p and UCP2 at the middle stage during ovine preadipocytes differentiation.In conclusion,miR-132-3p inhibits UCP2 expression at both levels of mRNA and protein by specifically binding to UCP2 3'-UTR,and thus promotes ovine preadipocytes differentiation to a certain extent.