Identification And Function Study Of Mirna Associated With Porcine Intramuscular Fat Deposition

Nowadays, with improvement of living quality, consumption demands have changed from quantity to quality, whereby more attention is paid to the taste, juiciness and tenderness of pork. Intramuscular fat (IMF), or "marbling", is a crucial trait that influences pork quality. Hence, under the premise of lean meat, appropriate increasing IMF content to improve the meat quality has become a new hotspot of research. IMF content is determined by the growth and development of adipocytes. Previous studies on IMF mainly focused on the conversion of preadipocytes to adipocytes, adipocyte lipid metabolism and promoting adipogenesis within skeletal muscle. Although huge efforts have been exerted to enhance IMF content in pig only very limited success has been achieved, while the detailed mechanism of IMF accumulation in pigs is still unknown. In this study we used high-throughput sequencing methods, and preliminary screened some miRNAs, genes and SNPs associated with intramuscular fat deposition in pigs. Our study identified that PGK1and CXHXorf23were miR-365-3p’s target genes, moreorer miR-365-3p may affect C2C12cells induced adipogenic differentiation。Our study may lay a foundation to explore the molecular deposition mechanism of IMF in skeletal muscle. The main results as follows:(1) We first identified the expression of miRNAs and genes in American Large White by comparing longissimus dosi with different IMF content DGE data, and found44miRNAs and129genes. Among these genes, we verified8miRNAs (7miRNAs were up-regulated,1was down-regulated), and4genes by Real-Time PCR. The results were consistent with the sequencing.(2) We performed a GWAS study in233castrates United States Department of Large White resoureds for IMF content trait. We found42significant SNPS associated with IMF content trait, whereby15of them were located on chromosome14.(3) We examined the expression of miR-365-3p, PGK1and CXHXorf23in several tissues (including heart, liver, spleen, lung, kedney, small intestine, stomach, leg muscle, longissimus dorsi, lymph and fat) from American Large White by Q-PCR. (4) We induced C2C12cells into fat, and maintained them in special medium for collecting RNA in various time points (0day,1days,2days and4days), We tested miR-365-3p, PGK1and CXHXorf23gene’s expression level by Q-PCR. It showed that the expression of miR-365-3p and PGK1gene were up-regulated, CXHXorf23gene was down-regulated.(5) We fused swine PGK1and CXHXorf23’s3’UTR to a duo-luciferase reporter gene plasmid(psiCHECKTM-2vector) and miR-365-3p mimics,then induced into cells to verify whether miR-365-3p could bind to putative site of PGK1or CXHXorf23mRNA’s3’UTR. We found that luciferase activity was significantly decreased when miR-365-3p overexpressed.(6) By overexpression of miR-365-3p when C2C12cells were induced into fat and collecting RNA in different time points (0day,1day,2days and4days), we tested the two types of fat maker genes, C/EBPa and FABP4mRNA expression level. The result showed that C/EBPa and FABP4mRNA expression level were significantly up-regulated at4days.