| Adipocyte differentiation and fat deposition are vital factors affecting the growth and development of cattle and beef quality,and are also the technical focus and difficulty of close attention in modern beef cattle breeding and production.miRNAs,an important class of genetic regulators,regulate the expression and function of candidate genes for muscle development and fat differentiation traits in ruminants at the post-transcriptional level by binding to target mRNAs.Currently,some studies have shown that miRNAs were associated with adipocyte differentiation and adipocyte function.Previously,the laboratory also obtained adipocyte differentiation candidate differentially expressed miRNAs(miR-145 and others)by screening and analyzing bovine preadipocytes and mature adipocytes differentially expressed miRNAs.In this study,miR-145,which was specifically and highly expressed in bovine preadipocytes,was used as the target.Synthesis of miR-145 mimics and inhibitors,analysis and validation of the effects of miR-145 on proliferation,differentiation and lipid metabolism of bovine preadipocytes.Validation of the target relationship of miR-145 with the candidate gene INSIG1 by bioinformatic prediction and analysis of target genes using a dual luciferase reporter.We further constructed an incremental expression vector and RNA interference vector for the target gene INSIG1 to further elucidate the role of INSIG1 gene in lipogenic differentiation and lipid metabolism-related pathways in bovine preadipocytes.The results of the obtained studies showed that:1.miR-145 was highly conserved and highly expressed in bovine preadipocytes.The target genes of miR-145 were predicted to be enriched in cell differentiation biological processes and lipometabolism signaling pathway,suggesting that miR-145 could regulate lipid metabolism and cell differentiation in bovine preadipocytes.2.As identified by oil red O staining,BODIPY staining,triglyceride,cholesterol and glycerol content determination,Ed U and qPCR analysis,miR-145 inhibited the proliferation of bovine preadipocytes,promoted the differentiation of bovine preadipocytes,increased the content of triglyceride,cholesterol and glycerol in cells and the accumulation of lipid droplets,and had an effect on fatty acid content.3.The predicted target genes were verified by qPCR at the mRNA level,and four functional genes were screened for negative regulatory relationships with miR-145,KLF14,AGPAT3,INSIG1,and CDK6.Dual luciferase reporter vectors for the above four candidate genes were successfully constructed.The dual-luciferase reporter analysis determined that miR-145 had a target relationship with INSIG1,and the negative regulatory relationship of miR-145 with INSIG1 was verified at the mRNA level and protein level,respectively.4.INSIG1 was highly expressed in liver and adipose tissue of cattle.INSIG1 inhibited the differentiation of bovine preadipocytes,decreased the contents of triglyceride,cholesterol,glycerol and lipid droplet accumulation,and affected the content of fatty acid.5.Both miR-145 and INSIG1 could affect the expression of PPARγ,a key gene for lipogenic differentiation,and thus regulate the process of lipogenic differentiation in bovine preadipocytes.In addition,the effect of INSIG1 on the negative regulation of PRKAA1 gene mRNA expression levels suggested that INSIG1 may be involved in the AMPK signaling pathway,which regulates lipid metabolism in bovine precursor adipocytes.In summary,bovine miR-145 could target and negatively regulated INSIG1 gene expression in adipocyte lipogenic differentiation and lipid metabolism.The results of the study provided a technical basis for screening key genes and regulatory elements of bovine preadipocytes proliferation and adipocyte differentiation,and resolving their regulatory roles in the pathway and molecular mechanisms. |
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