miR-33a靶向Lipin1和IRS2调节绵羊前体脂肪细胞分化的研究

旨在揭示miR-33a在绵羊前体脂肪细胞分化中的生物学功能。本研究以15日龄雄性绵羊背部皮下前体脂肪细胞为试验材料，所有的试验均设立3个重复；利用生物信息学软件预测miR-33a的靶基因，并通过双荧光素酶报告试验对预测的潜在靶基因进行验证；用qPCR和Western blotting分别检测miR-33a、Lipin1和IRS2及其编码蛋白的表达，以揭示miR-33a与其靶基因在绵羊前体脂肪细胞分化中的表达规律；慢病毒介导实现miR-33a的过表达和干扰后，检测Lipin1、IRS2和成脂标志基因的表达，并用油红O染色检测脂滴沉积能力，以解析miR-33a对其靶基因的调节机制。生物信息学分析发现，miR-33a与Lipin1和IRS2 3'-UTR都存在结合位点，miR-33a显著下调Lipin1和IRS2野生型双荧光质粒的相对荧光活性（P<0.05）；在绵羊前体脂肪细胞分化中，miR-33a与Lipin1和IRS2的表达趋势相反；过表达miR-33a后，显著下调了Lipin1（P<0.01）和IRS2（P<0.05）及其编码蛋白以及成脂标志基因的表达；干扰miR-33a后，这些基因和蛋白的表达则显著上调；过表达miR-33a减少了脂滴沉积，干扰miR-33a促进了脂滴沉积。在绵羊前体脂肪细胞分化中，miR-33a与Lipin1和IRS2的表达呈负相关。miR-33a靶向Lipin1和IRS2的3'-UTR抑制绵羊前体脂肪细胞分化和脂滴沉积。

miR-33a Regulates Ovine Preadipocyte Differentiation by Targeting Lipin1 and IRS2

The aim of this study was to reveal the biological functions of miR-33a during the differentiation of ovine preadipocytes. In this study, preadipocytes from the back fat of 15-day-old male lambs were used as research material. All the experiments in this study had 3 replicates. Bioinformatics software was used to predict the target genes of miR-33a. The potential target genes were verified by double luciferase reporter system. In order to reveal the expression pattern of miR-33a and its targeting genes during the differentiation of ovine preadipocytes, qPCR and Western blotting were used to detect the expressions of miR-33a, Lipin1, and IRS2 and proteins. In order to elucidate the regulatory mechanism of miR-33a on its target genes, miR-33a was overexpressed or interfered using lentivirus-mediated method. The expressions of Lipin1, IRS2 and adipogenic marker genes in ovine preadipocytes were detected. The lipid droplet deposition ability was measured by Oil Red O staining. The binding sites of miR-33a with 3'-UTR of Lipin1 and IRS2 were found using bioinformatics prediction. miR-33a significantly down-regulated the relative fluorescence activity of Lipin1 and IRS2 wild-type double fluorescent plasmids (P<0.05). The expression levels of miR-33a and Lipin1, IRS2 exhibited opposite trends during ovine preadipocyte differentiation. The overexpression of miR-33a significantly down-regulated the expressions of Lipin1 (P<0.01), IRS2 (P<0.05) and their encoded proteins, and adipogenic marker genes. After the miR-33a was interfered, the expressions of these genes and proteins were significantly up-regulated. Overexpression of miR-33a reduced lipid droplet deposition, and interference of miR-33a promoted lipid droplet deposition. In conclusion, the expressions of miR-33a and Lipin1, IRS2 were negative correlated during ovine preadipocyte differentiation. miR-33a negatively regulates the differentiation of ovine preadipocytes and the lipid droplets deposition by targeting the 3'-UTR of Lipin1 and IRS2.