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UCP2mRNA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UCP2mRNA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 inves- tigate 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 31-UTR,,and significantly down- regulated 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 pro-tein 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 3t-UTR, and thus promotes ovine preadipocytes differentiation to a certain extent.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2017年第11期2059-2067,共9页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家自然科学基金(31372292)
山西省优势肉用家畜高效安全生产协同创新中心项目
