miR-150靶向AOC3调控绵羊前体脂肪细胞分化的研究

miR-150 Regulates Ovine Preadipocyte Differentiation by Targeting AOC3

旨在通过探究miR-150与AOC3的靶标关系,揭示miR-150在广灵大尾羊前体脂肪细胞分化过程中的作用机制。本研究以3只5日龄发育正常且健康的广灵大尾羊公羊为试验材料,采用qPCR方法检测广灵大尾羊皮下、肾周和尾部脂肪组织中miR-150的表达量;采集广灵大尾羊的尾部脂肪组织并分离绵羊前体脂肪细胞;通过细胞免疫荧光技术鉴定细胞纯度;利用生物信息学软件预测和双荧光素酶报告试验验证miR-150与AOC3的靶标关系;转染miR-150 mimics和inhibitors并检测AOC3和成脂分化标志基因的表达情况;构建并转染绵羊AOC3基因的过表达和干扰载体至绵羊前体脂肪细胞,利用qPCR和Western blotting检测miR-150与AOC3在广灵大尾羊前体脂肪细胞分化中的表达规律;油红O染色检测成脂能力,上述试验方法均设立3个生物学重复。结果表明,miR-150在3个不同部位脂肪组织中尾部脂肪表达量最高,差异极显著(P<0.001)。与对照组相比,过表达miR-150后,AOC3基因和成脂标志基因的表达显著下调(P<0.05),且脂滴生成减少,抑制了绵羊前体脂肪细胞分化;抑制miR-150后,结果相反。miR-150与AOC3的3′-UTR存在结合位点,miR-150极显著降低了AOC3野生型的相对荧光活性(P<0.001)。过表达AOC3后,成脂分化标志基因的表达极显著上调(P<0.01),脂肪细胞产生了更多脂滴,AOC3促进了绵羊前体脂肪的分化;干扰AOC3后,结果相反。本研究表明,miR-150与AOC3的3′-UTR存在结合位点,在绵羊前体脂肪细胞分化的过程中,miR-150与AOC3的表达量呈负相关性,miR-150通过靶向AOC3抑制脂肪分化,研究结果为microRNAs(miRNAs)在分子水平上的研究提供了一定的理论依据。

The aim of this study was to reveal the action mechanism of miR-150 during the differentiation of preadipocytes in Guangling Large-Tailed sheep by investigating the relationship between miR-150 and target gene AOC3.In this study,three 5-day-old Guangling Large-Tailed male sheep with normal development and health were used as the research material.The expression level of miR-150 in subcutaneous,perirenal and tail adipose tissues of Guangling Large-Tailed sheep was detected by qPCR.Tail adipose tissue of Guangling large-tailed sheep was collected to isolate ovine preadipocytes.Cell purity was determined identified by cell immunofluorescence.Bioinformatics software was used to predict the target relationship between miR-150 and AOC3,and double luciferase reporter system was used to verify this relationship.miR-150 mimics and inhibitors were transfected into preadipocyte and the expression of AOC3 and adipogenic marker genes were detected.The overexpression and interference vector of ovine AOC3 were constructed and transfected into ovine preadipocytes.qPCR and Western blotting were used to detect the expression patterns of miR-150 and AOC3 during the differentiation of preadipocytes in Guangling Large-Tailed sheep.Lipid accumulation ability was detected by Oil Red O staining.Three biological replicates were set up for each of the above tests.The results showed that the expression of miR-150 was the highest in tail fat,and the difference was significant(P<0.001).After overexpression of miR-150,the expression of AOC3 and adipogenic marker genes were significantly down-regulated(P<0.05),lipid droplet accumulation decreased.These results implied that miR-150 inhibits the differentiation of ovine preadipocytes.The results were opposite after inhibition of miR-150.There was a binding site between miR-150 and the 3′-UTR of AOC3,and miR-150 was extremely significant decreased the relative fluorescence activity of wild type AOC3(P<0.001).After overexpressing AOC3,the expression of adipogenic marker genes were significantly up-regulated(P<0.01)and more lipid droplets were produced,indicating that AOC3 promotes the differentiation of ovine preadipocytes.After interfering with AOC3,the result was reversed.In conclusion,there is a binding site between miR-150 and the 3′-UTR of AOC3.The expression level of miR-150 was negatively correlated with that of AOC3 during the differentiation of ovine preadipocyte.miR-150 inhibits the differentiation of ovine preadipocytes by targeting AOC3.The results provide a theoretical basis for the research of microRNAs(miRNAs)at the molecular level.