Identification Of MiRNAs And Their Target Genes In Adipose Tissue Of Different Pigs And Bioinformatics Analysis Of Ssc-miR-486

The domestic pig, is not only an important species in agricultural economy, but also a good model organism for medical research. In order to investigate the genetic regulators that could control fat deposition, Laiwu pig and Large White pig were selected. They are quite different in terms of fat deposition capability. Two small RNA libraries respectively from subcutaneous adipose tissue of Large White pig (D) and subcutaneous adipose tissue of Laiwu pig (L) were constructed. Then the expression profiles of miRNAs in the adipose tissue derived from two different pigs were determined using RNA-Seq to identify miRNAs that might regulate fat deposition. Moreover, we carried out a genome-wide analysis and prediction of these miRNAs and their target genes.miR-486 was first identified in human fetal liver tissue. Currently, function of this miRNA was reported mainly in terms of cancer and muscle. However, the study showed significant upregulation of miR-486 in the adipose tissue of Laiwu pigs, indicating that ssc-miR-486 involved in the regulation of fat deposition in pigs. Thus, the real-time PCR was applied to verify differential expression of ssc-miR-486 in the adipose tissue of Laiwu pig and Large White pig. Then we performed bioinformatics analysis of ssc-miR-486, including the prediction of the target genes and transcription factor binding sites, the analysis of sequence conservation and Gene Ontology (GO)and KEGG pathway of its target genes using the databases (miRBase, Ensembl and NCBI) and bioinformatic softwares (miRanda, PITA, RNAhybrid, Cytoscape and JASPAR) to explore its biological roles in the pocine adipose tissue.The major results based on investigation above are as follows:1. A total of 8259953 and 8553739 clean reads from L and D were respectively obtained. The maximum peak of sequence length distribution occurs around 22nt, in keeping with the features of the miRNA length. Further analysis indicated that compared with Large White pig,17 known miRNAs, including ssc-miR-133a-3p, ssc-miR-486 and ssc-miR-204, were up-regulated in Laiwu pig, and 22 known miRNAs, including ssc-let-7i and ssc-miR-27b-3p were down-regulated in Laiwu pig. In addition, we identified 55 differentially expressed novel miRNAs between Laiwu pig and Large White pig.2.GO and KEGG analysis revealed that the predicted targets of these differentially expressed miRNAs were significantly enriched in several fat-associated pathways such as PPAR, MAPK and Wnt signaling pathways. In addition, for the first time we found that three miRNAs, including ssc-miR-133a-3p, ssc-miR-486 and ssc-miR-1, may respectively target genes CPT1A, SCD5 and SCD in the PPAR signaling pathway and change its signal transduction, thus playing a role in regulating the porcine fat deposition.3. In the second part of the experiment, the target gene prediction and bioinformatics analysis of ssc-miR-486, which was identified as an upregulated miRNA in Laiwu pig, were performed. First, the result of qPCR showed ssc-miR-486 was indeed significantly upregulated in the adipose tissue of Laiwu pig which has a stronger ability of accumulating fat. The bioinformatics analysis showed that miR-486 was very conservative among various species and multiple transcription factor binding sites, such as SREBP1 and SREBP2, were found in the promoter area of ssc-miR-486. Additionally,229 target genes of ssc-miR-486 were significantly enriched in many biological processes, for example, lipid metabolism, lipid biosynthesis and cell metabolism etc. and signal transduction pathways such as steroid biosynthesis, inositol phospholipid metabolism and PPAR signaling pathway. The results suggest that ssc-miR-486 play a certain role in porcine fat deposition and lipid metabolism and provide a basis for fat deposition research in the future.In a word, our results provide a comprehensive analysis of miRNAs related with fat deposition and the biological role of ssc-miR-486. These lay the theoretical basis for the cultivation of fine pig breeds and treatment of human obesity-related diseases.