Small RNAomics Of Subcutaneous Fat And The Role Of MiR-124-3p In Preadipocyte Differentiation Of Sheep

MicroRNA(miRNA)is a kind of short-chain non-conding RNAs.MiRNAs can binding to the mRNA of coding genes to inhibit or degrade the targeting mRNA,and play roles in regulating the development and metabolism of various kind of tissues and organs.In this way,miRNAs play roles in mediating the differentiation,fat deposition,and metabolism in adipose tissue.Recent study mainly focused on the effect of miRNA on model animal development,with less investigation on the regulatory rules in ovine adipse tissue.The present study mainly pays attention in the function and molecular mechanism during ovine adipocyte differentiation and fat deposition,thus providing theory foundations for improving the adipose distribution and carcass quality.In order to investigate the roles of miRNA in adipose development of sheep,high-throughput sequencing was used to detect the expression of miRNA in subcutaneous fat of Shanxi Meat Sheep Dam Line(SMD)and Small-tailed Han Sheep(STH).Stem-loop qPCR was used to validate the accuracy of miRNA-seq.Know miRNAs and novel miRNAs in two libraries were identified using bioinformatics methods,and their differential expression between two libraries were calculated.Besides,the targets of differential expressed miRNA were predicted and classified to various functions using Gene Ontology and KEGG.MiR-124-3p as a differential expressed miRNA in two libraries was selected to verify its roles in adipogenesis of adipose-derived and skeletal muscle-derived preadipocytes.Dual-luciferase reporter assay was used to validate the binding relationship between miR-124-3p and C/EBPa or BCKDHA.Furthermore,the lentivirus expression and block system for BCKDHA was established and used to infect the preadipocytes.After lentivirus infection,the branched-chain amino acid consumption and adipogenesis ability of muscle-derived preadipocytes was observed.The main results obtained in the present study are showing below.The first,through high-throughput sequencing for the miRNA expression in subcutaneous fat of sheep,11,264,198 and 14,715,022 clean reads were characterized in STH and SMD libraries,respectively.The clean reads were mapped to 121 and 119 known,along with 163 and 184 novel miRNAs in two libraries.Differential expression analysis result shows that 43 known miRNA and 117 novel miRNAs were significantly differential expression in two libraries,corresponding to 297,964 and 425,278 potential target genes.In order to validate the accuracy of high-throughput sequencing,the expression of miR-103,miR-107,miR-124-3p,miR-191,miR-221,novel-miR-42 and novel-miR-121 was detected in subcutaneous fat of STH and SMD.Results showed that the tendency of two methods were corresponded and indicated the high-throughput sequencing was in sharp.The second,miR-124-3p as a differentially expressed miRNA was seleted to search its roles in adipogenic differentiation and the underlying mechanism.Bioinformatic methods were used to predict the target genes of miR-124-3p,and results showed that C/EBPa and BCKDHA mRNA 3'-UTR have binding sites with the seed region of miR-124-3p.Dual-luciferase reporter assay validated the predicted results.Ovine adipose and skeletal muscle-derived preadipocytes were isolated and cultured in vitro.After overexpression the miR-124-3p in preadipocytes,the differentiation process of preadipocytes was slowed down and decreased the formation of lipid droplets.Besides,the mRNA and protein expression of C/EBPa,BCKDHA,and adipogenic associated marker genes were decreased with the treatment of miRNA overexpression.The third,lentivirus overexpression or inhibition system for BCKDHA was established.After stimulating the expression of BCKDHA,the adipogenesis ability of muscle-derived preadipocytes was enhanced,along with the increased branched-chain amino acid consumption for culture medium.The block of BCKDHA expression in preadipocytes showed the converse results.Results demonstrating the BCKDHA induced branched-chain amino acid metabolism was essential for the adipogenesis of ovine muscle-derived preadipocytes.The present study identified the functional miRNAs in regulating adipose metabolism using high-throughput sequencing and validated the specific roles of miR-124-3p in adipose metabolism.Reference foundations will be provided for further investigating the roles of miRNAs in adipogenesis for domestic animals.