Identification of novel regulators in porcine skeletal muscle growth by integrated analysis of miRNA and mRNA expression

In order to discover novel regulatory pathways of porcine muscle development, integrated analysis of microRNA and mRNA expression data were conducted using day 33, 65, 90 gestation fetal and adult Landrace longissimus muscle samples. Our results showed that 1,947 out of 20,201 genes and 214 out of 779 microRNAs are statistically differentially expressed at the 4 ages, which involved in 35 pathways. Further, a conjoint analysis refined the corresponding regulatory relationship between microRNAs and mRNAs based on pair-wised comparisons. The top 10 enrichment microRNAs were selected to match differentially expressed genes which were considered to be responsible for myogenesis. To gain additional insight into the mechanisms underlying the genes regulated by its microRNAs, the gene ontology (GO) analysis was conducted for each pair-wised comparison. The results showed that myofibril term (GO:0030016, 0043292), sarcomere term (GO:0030017) and contractile fiber (GO:0044449) term were enriched at early stage (E33), which implied microRNAs involved in the refinement process of myogenesis of porcine skeletal muscle. Further in vitro functional assays confirmed microRNAs mediated regulation for some genes in the above pathways. MiR-214 and miR-133 were chosen for in vitro functional assay respectively. The results showed that miR-214 can increase C2C12 cell proliferation, and the gain and loss of function studies showed miR-133b could promote C2C12 cell differentiation and repress cells proliferation by targeting MAPK pathway genes. Over-expression of miR-133b repressed phosphorylation of ERK1/2 during myoblasts differentiation. Our work offered novel information on microRNAs mediated myogenesis and muscle development.