Study On Molecular Mechanism Of Inhibiting Bovine Preadipocytes Differentiation And Promoter Activity Of MiR-145

The proportion of "marble" pattern is an important factor affecting beef quality and one of the important objectives of beef cattle breeding.At the molecular level,the regulation of differentiation of bovine adipocytes can provide a strong theoretical basis for beef cattle breeding.Micro RNAs(miRNAs)are a class of non-coding short-fragment RNAs that target and inhibit the expression of functional genes through post-transcriptional regulatory mechanisms,and are thus widely involved in various cell life activities.Since 2004,miR-143 has been found to promote adipocyte differentiation,and then more and more miRNAs have been found to be involved in the regulation of adipocyte differentiation.MiR-145,classified into the same cluster with miR-143,has been shown to have the characteristics of specific high expression in adipose tissue and differential expression during adipocyte differentiation in multiple published studies.However,there are few studies on the regulatory role and regulatory network of miR-145 in adipocyte differentiation.In this study,Qinchuan cattle preadipocytes were used as the research materials to explore the potential mechanism of miR-145 upstream and downstream regulatory network in the differentiation of bovine adipocytes.Firstly,the expression patterns of miR-145 in Qinchuan cattle tissues and the differentiation process of preadipocytes was determined by qRT-PCR method.It was confirmed that miR-145 may be involved in the regulation of bovine adipogenic differentiation.Then,the effects of miR-145 on the differentiation of bovine preadipocytes were explored by means of adenovirus-mediated gene function acquisition and deletion methods.Meanwhile,in combination with RNA-seq sequencing analysis and target prediction analysis,this study further clarified the potential major pathways and key targets regulated by miR-145 in bovine preadipocytes,and preliminarily verified the molecular mechanism of miR-145 involved in the differentiation regulation of bovine preadipocytes through dual-luciferase activity analysis and western blot analysis.Finally,this study also explored the upstream transcription factor that regulates bovine miR-145 gene expression by means of 5’RACE experiment,integration analysis of CHIP-seq data,prediction of transcription factor binding site,dual-luciferase activity analysis and EMSA experiment.The main research results are as follows:(1)The miR-145 gene was widely expressed in various tissues of Qinchuan cattle,especially in the longissimus dorsi,heart,subcutaneous fat,liver and perinenal fat.Moreover,during the differentiation of bovine preadipocytes,miR-145 showed a downregulation trend,and the expression level was the lowest on the eighth day of differentiation.Based on the above results,this study selected miR-145 as a candidate miRNA for the regulation of bovine preadipocytes differentiation.(2)A stable recombinant adenovirus overexpression vector with bovine miR-145 precursor sequence as the insertion fragment and a stable recombinant adenovirus interference vector with 8×sponge of the bovine miR-145 gene mature sequence as the insertion fragment were constructed.Overexpression and interference experiments confirmed that miR-145 could significantly inhibit the differentiation of bovine preadipocytes,but had no significant effect on proliferation and apoptosis.These results indicated that miR-145 mainly played a negative role in regulating differentiation in the process of life activities of bovine preadipocytes.(3)The RNA-seq results showed that the differentially expressed genes caused by overexpression and interference with miR-145 in the bovine preadipocytes were mainly enriched in the PI3K/Akt pathway and the MAPK pathway,which was consistent with the enrichment results of the predicted targets of miR-145.In addition,key genes of PI3K/Akt pathway(IRS1)and MAPK pathway(NRAS)and their common upstream gene IGF1 R were confirmed to be targets of miR-145.(4)During the differentiation of bovine preadipocytes,miR-145 not only significantly inhibited the protein expression of IGF1 R,IRS1 and NRAS genes,but also significantly inhibited the phosphorylation of Akt and ERK1/2 proteins.As an activator of the PI3K/Akt pathway and the MAPK pathway,insulin can rescue the downregulation of Akt and ERK1/2protein phosphorylation levels caused by overexpression of miR-145.(5)The transcription initiation site(TSS)of bovine miR-145 gene was determined to be located at 1353 bp upstream of this gene by 5’RACE.Through integration analysis of CHIP-seq data and prediction of transcription factor binding site,it was found that there were conserved potential C/EBPβ binding sites at 1954 bp(-601)and 2231 bp(-878)upstream of bovine miR-145 gene.(6)Downregulation of C/EBPβ expression resulted in significantly increased miR-145 expression level and miR-145 promoter activity.Detection of promoter region deletion vector activity indicated that the potential C/EBPβ binding site at-601 affected the transcriptional activity of bovine miR-145 gene promoter.EMSA assay further verified the binding of C/EBPβ to the promoter region of bovine miR-145 gene.These results showed that transcription factor C/EBPβ targeted to the promoter region of bovine miR-145 gene and was a negative regulator of bovine miR-145 gene expression.In conclusion,this study from the point of miR-145 negative effect on the differentiation phenotype of bovine preadipocytes,it was found that miR-145 targeted and inhibited IGF1 R,IRS1 and NRAS,and down-regulated the phosphorylation levels of PI3K/Akt pathway and MAPK pathway.These regulatory relationships may be the major cause for miR-145 inhibiting the differentiation process of bovine preadipocytes.In addition,this study also identified the transcription initiation site of bovine miR-145 and confirmed that C/EBPβ,a key transcription factor for adipocyte differentiation,is a transcription inhibitor of bovine miR-145.This study preliminarily discussed the upstream and downstream regulation relationships of miR-145 in bovine preadipocytes,systematically explored the role and positioning of miR-145 in the regulation network of adipose differentiation,laying a theoretical foundation for further studies.