Study On Function Of MiR-148a And The Reference Genes For QPCR In Skeletal Muscle Development

This paper focused on two topics in skeletal muscle development:the miR-148a function and its molecular mechanism in skeletal muscle development; the selection of reference genes for the normalization of qPCR gene expression in porcine skeletal muscle at different development stages. The main work and results were as following:1. Study of miR-148a function in skeletal muscle developmentMicroRNA (miRNA) are a class of evolutionarily conserved and noncoding small RNAs. They post-transcriptionally regulate gene expression through mRNAs degradation or translational repression. Thus, they have important roles in many biological processes including early development, cell proliferation, differentiation and apoptosis.Our previous microRNA transcriptome profiling analysis, using a Solexa deep sequencing approach revealed a series of differentially expressed miRNAs at different developmental stages of pig embryonic and adult skeletal muscle. We found that miR-148a was highly expressed in embryonic skeletal muscle tissue, but not expressed in the adult skeletal muscle. The miR-148a has been reported to be correlated to cancer pathogenesis, but there has been no report about its function in skeletal muscle development so far.In our study, we identified the function of miR-148a in skeletalmuscle development. We identified miR-148a promoted myogenic differentiation and enhanced cell cycle arrest. We also found miR-148a positively regulated myogenic differentiation via ROCK1downregulation.(1) We first established a C2C12cells differentiation model in vitro and analyzed the expression of miR-148a in C2C12cells cultured in growth medium or differentiation medium (DM) for0,1,3or5d by qPCR. We found that the expression of miR-148a increased during C2C12differentiation.(2) We overexpressed miR-148a in C2C12and employed microarray analysis to identify differentially expressed genes. Some muscle-fiber genes were significantly up-regulated according to the microarray data. And we verified six of the up-regulated genes (Tnnt3, Mb, Mylpy, Myom3, myogenin and IGF2) by using qPCR.(3) We overexpressed or inhibited miR-148a in C2C12and primary muscle cells and induced the differentiation. Through the detection of myogenic marker genes (MHC, myogenin and Skeletal a-actin) expression changes in mRNA or protein leves, we found that overexpression of miR-148a enhanced myogenic marker genes expression and the inhibiton of miR-148a decreased the mRNA and protein expression of myogenic markers, indicating miR-148a was an essential positive regulator of myogenesis and accelerated myogenic differentiation.(3) We also analyzed the role of miR-148a in C2C12proliferation by using CCK-8Cell Counting Kit, but we were unable to detect any miR-148a-dependent effects on cell proliferation after the overexpression or inhibiton of miR-148a in C2C12. However, the FACS profile for DNA content demonstrated that transfecting miR-148a increased the Gl phase population by10%(P<0.05) and decreased the S phase population of C2C12myoblast cells, indicating that it promoted cell cycle quiescence.(4) Through computational target prediction programs (TargetScan and miRanda), we identified Rho-associated coiled-coil containing protein kinase1(ROCK1) as the potential target of miR-148a. ROCK1is a known inhibitor of myogenic differentiaton. A dual-luciferase reporter assay was used to demonstrate that miR-148a directly targeted the3'UTR of ROCK1. In addition, by using qPCR and western blot, we found that miR-148a directly targeted the ROCK1gene by translational repression without affecting mRNA levels.(6) We used siRNA to inhibit ROCK1expression in C2C12and primary muscle cells.The results showed ROCK1siRNA significantly inhibited ROCK1expression but accelerated myogenic marker genes expression, indicating that ROCK1was a negative regulator of myogenesis and the inhibiton of ROCK1promoted myogenic differentiation.2. The selection of reference genes for qPCR in porcine skeletal muscle at different development stages.In gene expression analysis, such as qPCR, reference genes are used to normalize the gene expression. The reference genes should be validated, ideally with consistent expression under various conditions. However, many studies prove that the expression of reference genes vary in different tissues, different cells and different development stages Thus, the selection of appropriate reference genes for qPCR data normalization is critical step to obtain accurate and reliable gene expression profiles.In this study, we attempt to select a set of reliable reference genes for the normalization of qPCR gene expression in porcine different tissues and skeletal muscle at different development stages. We first used qPCR to examine expression of six common reference genes (GAPDH, ACTB, H3F3A, HPRT1, RPL32, and RPS18) in adult tissues and prenatal skeletal muscles at33,65, and90days postcopulation (dpc) from Tongcheng and Landrace pigs. The expression stability of these reference genes was evaluated by NormFinder, BestKeeper and geNorm methods. Our data suggest that the reference genes were expressed variably in different tissues, developmental stages and breeds. However, RPS18, PRL32and H3F3A expressed more stably then other candidate genes.In this study, we also analyzed the combination of the obove three reference genes. During skeletal muscle development, H3F3A and RPS18would be the most appropriate combination to normalize gene expression in Tongcheng pigs, whereas the combination of PRL32and RPS18would be more suitable in Landrace pigs. In different tissues, the expression of PRL32and RPS18was the most consistent, and the combination of three genes (RPL32, RPS18and H3F3A) was the most suitable for accurate normalization. Therefore, this study will provide some more reliable reference gene combinations for qPCR of gene expression studies in pig tissues and skeletal muscle development.