miR-23b-3p通过靶向PDE4B基因调控山羊肌内前体脂肪细胞的分化

miR-23b-3p regulates the differentiation of goat intramuscular preadipocytes by targeting the PDE4B gene

本研究旨在明确miR-23b-3p对山羊肌内前体脂肪细胞分化的影响,并确定这种作用是通过靶向基因PDE4B来实现的。基于实验室前期转录组测序结果,以筛选得到的山羊肌内脂肪细胞分化前后差异表达的miR-23b-3p为切入点,利用实时荧光定量PCR(real-time quantitative-polymerase chain reaction,qPCR)技术检测miR-23b-3p在山羊肌内前体脂肪细胞分化过程中的表达模式,从形态学水平和分子水平确定miR-23b-3p对脂肪分化及脂肪分化标志基因的影响;利用生物信息学预测以及双荧光素酶报告基因试验确定miR-23b-3p的下游靶基因,明确miR-23b-3p与预测靶标基因的靶向关系。结果表明,过表达miR-23b-3p后山羊肌内脂肪细胞脂滴积聚减少,成脂标志基因AP2、C/EBPα、FASN、LPL表达水平极显著下调(P<0.01),C/EBPβ、DGAT2、GLUT4和PPARγ的表达水平显著下调(P<0.05)。干扰miR-23b-3p表达后,山羊肌内脂肪细胞中脂滴积聚增多,ACC、ATGL、AP2、DGAT2、GLUT4、FASN和SREBP1表达水平极显著上调(P<0.01),C/EBPβ、LPL和PPARγ的表达水平显著上调(P<0.05)。通过生物信息学分析预测,PDE4B可能为miR-23b-3p的靶标基因,且过表达miR-23b-3p后极显著降低PDE4B的mRNA表达水平(P<0.01),干扰miR-23b-3p后PDE4B的mRNA水平得到了显著提升(P<0.05)。双荧光素酶报告基因试验结果表明,miR-23b-3p与PDE4B基因存在靶向关系。miR-23b-3p通过靶向PDE4B基因调控山羊肌内前体脂肪细胞的分化。

This study aimed to explore the effect of miR-23b-3p on the differentiation of goat intramuscular preadipocytes,and to confirm whether miR-23b-3p plays its roles via targeting the PDE4B gene.Based on the pre-transcriptome sequencing data obtained previously,the miR-23b-3p,which was differentially expressed in goat intramuscular adipocytes before and after differentiation,was used as an entry point.real-time quantitative-polymerase chain reaction(qPCR)was used to detect the expression pattern of miR-23b-3p during the differentiation of goat intramuscular preadipocytes.The effects of miR-23b-3p on adipose differentiation and adipose differentiation marker genes were determined at the morphological and molecular levels.The downstream target genes of miR-23b-3p were determined using bioinformatics prediction as well as dual luciferase reporter assay to clarify the targeting relationship between miR-23b-3p and the predicted target genes.The results indicated that overexpression of miR-23b-3p reduced lipid droplet accumulation in goat intramuscular adipocytes,significantly down-regulated the expression levels of adipogenic marker genes AP2,C/EBPα,FASN,and LPL(P<0.01).In addition,the expressions of C/EBPβ,DGAT2,GLUT4 and PPARγ were significantly downregulated(P<0.05).After interfering with the expression of miR-23b-3p,lipid droplet accumulation was increased in goat intramuscular adipocytes.The expression levels of ACC,ATGL,AP2,DGAT2,GLUT4,FASN and SREBP1 were extremely significantly up-regulated(P<0.01),and the expression levels of C/EBPβ,LPL and PPARγ were significantly up-regulated(P<0.05).It was predicted that PDE4B might be a target gene of miR-23b-3p.The mRNA expression level of PDE4B was significantly decreased after overexpression of miR-23b-3p(P<0.01),and the interference with miR-23b-3p significantly increased the mRNA level of PDE4B(P<0.05).The dual luciferase reporter assay indicated that miR-23b-3p had a targeting relationship with PDE4B gene.MiR-23b-3p regulates the differentiation of goat intramuscular preadipocytes by targeting the PDE4B gene.