Identification Of Porcine Skeletal Muscle Related MiRNAs And Functional Study Of MiRNA-21during Skeletal Muscle Myogenesis

MicroRNAs are evolutionarily conserved small RNAs that post-transcriptionally regulate gene expression and have emerged as critical regulators of skeletal muscle development. MiRNAs have a stem-loop structure and target binding sites present in target genes; the combination of mature miRNA and nucleoprotein in the RNA-induced silence complex (RISC) can suppress the expression of target genes in three ways:degrading mRNA; suppressing the transcription of mRNA; and, strengthening messenger RNA(mRNA) deadenylation and accelerating degradation of mRNA. MiRNAs regulate the expression of hundreds of target mRNAs simultaneously, thereby controlling multiple cellular processes, including proliferation, differentiation, apoptosis, and myogenesis.Using Solexa deep sequencing, we detected229and209known miRNAs in swine skeletal muscle at90days prenatal (E90) and100days postnatal (D100), respectively. We identified138miRNAs that were up-regulated in muscle at E90and31miRNAs up-regulated at D100. Of these,9differentially expressed miRNAs were selected to validate the small RNA libraries by quantitative RT-PCR (QRT-PCR). We found that miRNA-21was down-regulated, with a17-fold change at D100(p<0.001). Here, we identified miR-21as a novel myogenic microRNA that mediated myogesis.Considering the dynamic expression profiles of miR-21during skeletal muscle development in pigs, we inferred that miR-21may exert its potential roles through targetting certain genes involved in skeletal muscle growth and development. Of the established research results showed that the miR-21as an original cancer inducer factor, has played positive roles in the carcinogenesis. However, what’s the function of miR-21in skeletal muscle in the process of the skeletal muscle development has not been reported. Our study used C2C12myoblasts trying to explore the function of miR-21during myogenesis. We confirmed that miR-21can significantly promote the myoblast proliferation and growth for the first time.Then, we predicted and identified one target genes of miR-21:TGFBI by using bioinformatics methods, and verified miR-21modulating the development of skeletal muscle through regulating TGFB/Akt-mToR signaling pathways preliminary.We firstly tested miR-21expression using total RNA isolated from C2C12myoblasts cultured in growth medium (GM)0d and differentiation medium (DM) for1,3and5d by quantitative RT-PCR assays. Then Immunofluorescence detection was applied to identify the effect of miR-21on C2C12myoblats proliferation, the results showed that48hours later, miR-21significantly promoted cell proliferation; characterized byexpression of mitosis marker genes-P-H3. Through computational target prediction programs (TargetScan and miRanda), we identified transforming growth factor induced gene (TGFBI) as the potential target of miR-21. A dual-luciferase reporter assay was used to demonstrate that miR-21directly targeted the3’UTR of TGFBI. In addition, the overexpression of miR-21decreased the protein expression of TGFBI, suggesting miR-21directly targeted the TGFBI gene at the translational level during differentiation. TGFBI is a known inhibitor of cell proliferation, and the inhibition of TGFBI via siRNA leads to accelerated proliferation progression. The FACS profile for DNA content demonstrated that transfecting siRNA increased the S phase population of C2C12myoblast cells, indicating that it promoted cell cycle, we detected TGFBI-dependent effects on cell proliferation in the CCK-8cell growth assay. Therefore, our results revealed a novel mechanism in which miR-21positively regulates myogenic via TGFBI downregulation.