Role In Skeletal Muscle Development Research Articles

Pax genes in myogenesis: alternate transcripts add complexity.

Pax3 and Pax7 are powerful myogenic inducers and hence play a critical role in skeletal muscle development and regeneration. In this paper we discuss the role of Pax3 and Pax7 in dorsal patterning of the somite with subsequent determination of myogenic precursor cells for muscle formation within the developing embryo and in adult muscle. Recent evidence of the ability of stem cells to contribute to muscle regeneration in adult tissues, and the role of Pax7 in conversion of multipotent stem cells to the myogenic lineage are also discussed. Several tissue specific Pax7 transcripts that encode isoforms with different DNA binding characteristics and potentially distinct transactivation specificities are identified. The expression of a range of transcripts in the determination of different tissue lineages and distinct cell populations both in the embryo and in the adult indicates an extraordinary level of complexity. A detailed understanding of these molecules and their functions during embryogenesis and adult muscle formation is imperative for future stem cell therapies.

MicroRNA-499-5p regulates porcine myofiber specification by controlling Sox6 expression

MicroRNAs (miRNAs) are a class of small non-coding RNAs that negatively regulate gene expression of target messenger RNAs (mRNAs) and miRNAs have been proven to play vital roles in skeletal muscle development. The miRNA-499-5p has been reported to be negatively related with the expression of Sox6, a critical transcription factor for the maintenance of fast-twitch skeletal muscle. In this study, we amplified a length of 2012-bp mRNA that contains a 1512-bp porcine Sox6 (pSox6) 3'UTR from skeletal muscle of a Duroc×Landrace×Yorkshire pig. By luciferase reporter assay we verified that pSox6 is a target of miR-499-5p. In extensor digitorum longus and Soleus muscles of pigs, the expression levels of miR-499-5p and pSox6 mRNA were also inversely correlated. Besides, overexpression of miR-499-5p in porcine satellite cells promoted the expression of MyHC I and MyHC IIa mRNA, along with a reduction of pSox6 mRNA. Taken together, these results indicate that miR-499-5p may facilitate the oxidative myofibers formation by downregulating pSox6 expression.

The lysine methyltransferase Ehmt2/G9a is dispensable for skeletal muscle development and regeneration.

BackgroundEuchromatic histone-lysine N-methyltransferase 2 (G9a/Ehmt2) is the main enzyme responsible for the apposition of H3K9 di-methylation on histones. Due to its dual role as an epigenetic regulator and in the regulation of non-histone proteins through direct methylation, G9a has been implicated in a number of biological processes relevant to cell fate control. Recent reports employing in vitro cell lines indicate that Ehmt2 methylates MyoD to repress its transcriptional activity and therefore its ability to induce differentiation of activated myogenic cells.MethodsTo further investigate the importance of G9a in modulating myogenic regeneration in vivo, we crossed Ehmt2floxed mice to animals expressing Cre recombinase from the Myod locus, resulting in efficient knockout in the entire skeletal muscle lineage (Ehmt2ΔmyoD).ResultsSurprisingly, despite a dramatic drop in the global levels of H3K9me2, knockout animals did not show any developmental phenotype in muscle size and appearance. Consistent with this finding, purified Ehmt2ΔmyoD satellite cells had rates of activation and proliferation similar to wild-type controls. When induced to differentiate in vitro, Ehmt2 knockout cells differentiated with kinetics similar to those of control cells and demonstrated normal capacity to form myotubes. After acute muscle injury, knockout mice regenerated as efficiently as wildtype. To exclude possible compensatory mechanisms elicited by the loss of G9a during development, we restricted the knockout within adult satellite cells by crossing Ehmt2floxed mice to Pax7CreERT2 and also found normal muscle regeneration capacity.ConclusionsThus, Ehmt2 and H3K9me2 do not play significant roles in skeletal muscle development and regeneration in vivo.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-016-0093-7) contains supplementary material, which is available to authorized users.

Regulation of Skeletal Muscle Myoblast Differentiation and Proliferation by Pannexins.

Pannexins are newly discovered channels that are now recognized as mediators of adenosine triphosphate release from several cell types allowing communication with the extracellular environment. Pannexins have been associated with various physiological and pathological processes including apoptosis, inflammation, and cancer. However, it is only recently that our work has unveiled a role for Pannexin 1 and Pannexin 3 as novel regulators of skeletal muscle myoblast proliferation and differentiation. Myoblast differentiation is an ordered multistep process that includes withdrawal from the cell cycle and the expression of key myogenic factors leading to myoblast differentiation and fusion into multinucleated myotubes. Eventually, myotubes will give rise to the diverse muscle fiber types that build the complex skeletal muscle architecture essential for body movement, postural behavior, and breathing. Skeletal muscle cell proliferation and differentiation are crucial processes required for proper skeletal muscle development during embryogenesis, as well as for the postnatal skeletal muscle regeneration that is necessary for muscle repair after injury or exercise. However, defects in skeletal muscle cell differentiation and/or deregulation of cell proliferation are involved in various skeletal muscle pathologies. In this review, we will discuss the expression of pannexins and their post-translational modifications in skeletal muscle, their known functions in various steps of myogenesis, including myoblast proliferation and differentiation, as well as their possible roles in skeletal muscle development, regeneration, and diseases such as Duchenne muscular dystrophy.

Physiology in Perspective: Why Do We Continue to Ignore Sex Differences?

In 2001, the Institute of Medicine (IOM) of the National Academy of Sciences issued a report in which they highlighted the importance of exploring sex differences in biomedical research and medical practice. The IOM report recognized the obvious biological fact that sex, as defined by the presence

Analysis of MicroRNA Expression Profiles in Weaned Pig Skeletal Muscle after Lipopolysaccharide Challenge.

MicroRNAs (miRNAs) constitute a class of non-coding RNAs that play a crucial regulatory role in skeletal muscle development and disease. Several acute inflammation conditions including sepsis and cancer are characterized by a loss of skeletal muscle due primarily to excessive muscle catabolism. As a well-known inducer of acute inflammation, a lipopolysaccharide (LPS) challenge can cause serious skeletal muscle wasting. However, knowledge of the role of miRNAs in the course of inflammatory muscle catabolism is still very limited. In this study, RNA extracted from the skeletal muscle of pigs injected with LPS or saline was subjected to small RNA deep sequencing. We identified 304 conserved and 114 novel candidate miRNAs in the pig. Of these, four were significantly increased in the LPS-challenged samples and five were decreased. The expression of five miRNAs (ssc-miR-146a-5p, ssc-miR-221-5p, ssc-miR-148b-3p, ssc-miR-215 and ssc-miR-192) were selected for validation by quantitative polymerase chain reaction (qPCR), which found that ssc-miR-146a-5p and ssc-miR-221-5p were significantly upregulated in LPS-challenged pig skeletal muscle. Moreover, we treated mouse C2C12 myotubes with 1000 ng/mL LPS as an acute inflammation cell model. Expression of TNF-α, IL-6, muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1) mRNA was strongly induced by LPS. Importantly, miR-146a-5p and miR-221-5p also showed markedly increased expression in LPS-treated C2C12 myotubes, suggesting the two miRNAs may be involved in muscle catabolism systems in response to acute inflammation caused by a LPS challenge. To our knowledge, this study is the first to examine miRNA expression profiles in weaned pig skeletal muscle challenged with LPS, and furthers our understanding of miRNA function in the regulation of inflammatory muscle catabolism.

FGF13 regulates proliferation and differentiation of skeletal muscle by down-regulating Spry1.

Generally, the secretory forms of FGF are known to regulate cell proliferation, differentiation and morphogenesis by binding to the extracellular domain of cell surface receptors. Intracellular FGFs (FGF11-14) are expressed principally in the nervous system. FGF13 is a microtubule-stabilizing protein that regulates neuronal polarization and migration. Previous studies have reported high expression of FGF13 in cultures of single muscle fibres. However, functions of FGF13 in muscle development have not been explored. Real-time RT-PCR was performed to detect expression of FGF13 during C2C12 muscle cell proliferation and differentiation. To further understand the role of FGF13, its effects on proliferation and differentiation were examined by western blot analyses of cells transfected with FGF13 siRNA or FGF13 expression plasmids, or treated with chemical MEK inhibitors. Effects of FGF13 on related signalling pathways in C2C12 cell proliferation and differentiation were determined. FGF13 inhibited C2C12 cell proliferation by up-regulating p27 mRNA level and by down-regulating Cyclin E protein expression, during cell proliferation. Additionally, FGF13 down-regulated Spry1 protein expression, activating the ERK1/2 pathway by phosphorylation and leading to C2C12 cell differentiation inhibition. Consequently, FGF13 seemed to function as a repressor of myoblast differentiation via the ERK1/2 pathway. Although FGF13 inhibited Spry1 regardless of cell proliferation or differentiation, its pathway activation occurred only during the stage of myoblast differentiation. FGF13 inhibited C2C12 cell proliferation and differentiation by down-regulating Spry1. These findings indicate that FGF13 played a negative regulatory role in skeletal muscle development.

Identification of miR-2400 gene as a novel regulator in skeletal muscle satellite cells proliferation by targeting MYOG gene

MicroRNAs play critical roles in skeletal muscle development as well as in regulation of muscle cell proliferation and differentiation. Previous study in our laboratory showed that the expression level of miR-2400, a novel and unique miRNA from bovine, had significantly changed in skeletal muscle-derived satellite cells (MDSCs) during differentiation, however, the function and expression pattern for miR-2400 in MDSCs has not been fully understood. In this report, we firstly identified that the expression levels of miR-2400 were down-regulated during MDSCs differentiation by stem-loop RT-PCR. Over-expression and inhibition studies demonstrated that miR-2400 promoted MDSCs proliferation by EdU (5-ethynyl-2′ deoxyuridine) incorporation assay and immunofluorescence staining of Proliferating cell nuclear antigen (PCNA). Luciferase reporter assays showed that miR-2400 directly targeted the 3′ untranslated regions (UTRs) of myogenin (MYOG) mRNA. These data suggested that miR-2400 could promote MDSCs proliferation through targeting MYOG. Furthermore, we found that miR-2400, which was located within the eighth intron of the Wolf-Hirschhorn syndrome candidate 1-like 1 (WHSC1L1) gene, was down-regulated in MDSCs in a direct correlation with the WHSC1L1 transcript by Clustered regularly interspaced palindromic repeats interference (CRISPRi). In addition, these observations not only provided supporting evidence for the codependent expression of intronic miRNAs and their host genes in vitro, but also gave insight into the role of miR-2400 in MDSCs proliferation.

Role of extracellular matrix in development of skeletal muscle and postmortem aging of meat

The integrity of skeletal muscle is maintained by the intramuscular connective tissues (IMCTs) that are composed of extracellular matrix (ECM) molecules such as collagens, proteoglycans, and glycoproteins. The ECM plays an important role not only in providing biomechanical strength of the IMCT, but also in regulating muscle cell behavior. Some ECM molecules, such as decorin and laminin, modulate the activity of myostatin that regulates skeletal muscle mass. Furthermore, it has been shown that decorin activates Akt downstream of insulin-like growth factor-I receptor (IGF-IR) and enhances the differentiation of myogenic cells, suggesting that decorin acts as a signaling molecule to myogenic cells. With animal growth, the structural integrity of IMCT increases; collagen fibrils within the endomysium associate more closely with each other, and the collagen fibers in the perimysium become increasingly thick and their wavy pattern grows more regular. These changes increase the mechanical strength of IMCT, contributing to the toughening of meat. However, in highly marbled beef cattle like Wagyu, intramuscular fat deposits mainly in the perimysium between muscle fiber bundles during the fattening period. The development of adipose tissues appears to disorganize the structure of IMCT and contributes to the tenderness of Wagyu beef. The IMCT was considered to be rather immutable compared to myofibrils during postmortem aging of meat. However, several studies have shown that collagen networks in the IMCT are disintegrated and proteoglycan components are degraded during postmortem aging. These changes in ECM appear to reduce the mechanical strength of IMCT and contribute to the tenderness of uncooked meat or cooked meat at low temperature. Thus, the ECM plays a multifunctional role in skeletal muscle development and postmortem aging of meat.

MicroRNA-128 targets myostatin at coding domain sequence to regulate myoblasts in skeletal muscle development

MicroRNAs (miRNAs or miRs) play a critical role in skeletal muscle development. In a previous study we observed that miR-128 was highly expressed in skeletal muscle. However, its function in regulating skeletal muscle development is not clear. Our hypothesis was that miR-128 is involved in the regulation of the proliferation and differentiation of skeletal myoblasts. In this study, through bioinformatics analyses, we demonstrate that miR-128 specifically targeted mRNA of myostatin (MSTN), a critical inhibitor of skeletal myogenesis, at coding domain sequence (CDS) region, resulting in down-regulating of myostatin post-transcription. Overexpression of miR-128 inhibited proliferation of mouse C2C12 myoblast cells but promoted myotube formation; whereas knockdown of miR-128 had completely opposite effects. In addition, ectopic miR-128 regulated the expression of myogenic factor 5 (Myf5), myogenin (MyoG), paired box (Pax) 3 and 7. Furthermore, an inverse relationship was found between the expression of miR-128 and MSTN protein expression in vivo and in vitro. Taken together, these results reveal that there is a novel pathway in skeletal muscle development in which miR-128 regulates myostatin at CDS region to inhibit proliferation but promote differentiation of myoblast cells.

Myocyte enhancer factor 2C function in skeletal muscle is required for normal growth and glucose metabolism in mice.

BackgroundSkeletal muscle is the most abundant tissue in the body and is a major source of total energy expenditure in mammals. Skeletal muscle consists of fast and slow fiber types, which differ in their energy usage, contractile speed, and force generation. Although skeletal muscle plays a major role in whole body metabolism, the transcription factors controlling metabolic function in muscle remain incompletely understood. Members of the myocyte enhancer factor 2 (MEF2) family of transcription factors play crucial roles in skeletal muscle development and function. MEF2C is expressed in skeletal muscle during development and postnatally and is known to play roles in sarcomeric gene expression, fiber type control, and regulation of metabolic genes.MethodsWe generated mice lacking Mef2c exclusively in skeletal muscle using a conditional knockout approach and conducted a detailed phenotypic analysis.ResultsMice lacking Mef2c in skeletal muscle on an outbred background are viable and grow to adulthood, but they are significantly smaller in overall body size compared to control mice and have significantly fewer slow fibers. When exercised in a voluntary wheel running assay, Mef2c skeletal muscle knockout mice aberrantly accumulate glycogen in their muscle, suggesting an impairment in normal glucose homeostasis. Consistent with this notion, Mef2c skeletal muscle knockout mice exhibit accelerated blood glucose clearance compared to control mice.ConclusionsThese findings demonstrate that MEF2C function in skeletal muscle is important for metabolic homeostasis and control of overall body size.

Contractile properties of slow and fast skeletal muscles from protease activated receptor‐1 null mice

Protease-activated receptors (PARs) may play a role in skeletal muscle development. We compared the contractile properties of slow-twitch soleus muscles and fast-twitch extensor digitorum longus (EDL) muscles from PAR-1 null and littermate control mice. Contractile function was measured using a force transducer system. Fiber type proportions were determined using immunohistochemistry. Soleus muscles from PAR-1 null mice exhibited longer contraction times, a leftward shift in the force-stimulation frequency relationship, and decreased fatiguability compared with controls. PAR-1 null soleus muscles also had increased type 1 and decreased type IIb/x fiber numbers compared with controls. In PAR-1 null EDL muscles, no differences were found, except for a slower rate of fatigue compared with controls. The absence of PAR-1 results in a slower skeletal muscle contractile phenotype, likely due to an increase in type I and a decrease in type IIb/x fiber numbers. Muscle Nerve 50: 991-998, 2014.

Identification of the STAC3 gene as a skeletal muscle-specifically expressed gene and a novel regulator of satellite cell differentiation in cattle.

Recent studies in mice and zebrafish suggest that the unannotated Src homology 3 and cysteine rich domain 3 (STAC3) gene plays an important role in skeletal muscle development and contraction. The objective of this study was to determine the tissue specificity of the bovine STAC3 gene and its potential role in the proliferation and differentiation of bovine satellite cells. The STAC3 mRNA was detected only in skeletal muscle among 18 bovine tissues examined by reverse transcription PCR. Western blotting revealed the expression of STAC3 protein in bovine skeletal muscle and the absence of it in 6 bovine tissues analyzed. Transfection of the bovine satellite cells with a pool of 2 STAC3 small interfering RNA (siRNA) caused a 90% reduction in STAC3 mRNA. Cell proliferation assays revealed that STAC3 knockdown had no effect on the proliferation rate of bovine satellite cells. Approximately 60% of bovine satellite cells transfected with STAC3 siRNA formed myotubes by 72 h of differentiation, whereas that percentage was 40% for those transfected with negative control siRNA (P < 0.05). At 24, 48, and 72 h of differentiation, bovine satellite cells transfected with STAC3 siRNA had greater mRNA expression of myogenin, myosin heavy chain 3, and myosin heavy chain 7, markers of myotubes, than those transfected with negative control siRNA (P < 0.05). These results suggest that the STAC3 gene is a negative regulator of the differentiation and fusion of bovine satellite cells into myotubes. However, STAC3 expression was increased during the differentiation of bovine satellite cells into myotubes. This suggests that STAC3 might have different functions in bovine myotubes than in bovine satellite cells.

Dynamic Expression of MicroRNA-127 During Porcine Prenatal and Postnatal Skeletal Muscle Development

MicroRNAs (miRNAs), evolutionarily conserved non-coding RNAs in length 21-24 bp, play a critical role in skeletal muscle development. In this study, to explore the function of mircoRNA-127 in porcine skeletal muscle development, eight tissue samples from adult pigs and longissimus muscle samples at 26 developmental stages were collected from Tongcheng and Landrace pigs. The spatial-temporal expression profiles of miRNA-127 were carried out using step-loop quantitative real-time PCR (stem-loop RT-PCR). To explore the molecular functions of miRNA-127, we predicted its target genes and performed functional annotation using bioinformatics methods. Results suggested that miRNA-127 was abundantly expressed in heart, ovary, uterus and spleen tissues and was weakly expressed in liver, lung, kidney and small intestine in both Tongcheng and Landrace pigs. And miRNA-127 showed significant expression differences in heart, ovary, spleen and uterus tissues between these two breeds. miRNA-127 basically kept at a relatively stable high level in middle and later embryonic stages and a low expression level in early embryonic stages and postnatal stages, but the expression levels of miRNA-127 were higher in Tongcheng pigs than in Landrace at most developmental stages. miRNA-127 potentially regulated 240 candidate genes. Results of Gene Ontology and KEGG pathway analysis indicated that these genes could be involved in many molecular functions and mechanisms, such as regulation of the force of heart contraction, regulation of transcription, regulation of T cell differentiation, MAPK signaling pathway and GnRH signaling pathway. Many significantly enriched GO terms and KEGG pathways were related to skeletal muscle development. This study will be helpful to understand the biological function for miRNA-127 and identify candidate gene associated with meat production traits in pigs.

Identification and profiling of microRNAs in the embryonic breast muscle of pekin duck.

MicroRNAs (miRNAs) regulate gene expression by fully or partially binding to complementary sequences and play important roles in skeletal muscle development. However, the roles of miRNAs in embryonic breast muscle of duck are unclear. In this study, we analyzed the miRNAs profiling in embryonic breast muscle of Pekin duck at E13 (the 13th day of hatching), E19, and E27 by high-throughput sequencing. A total of 382 miRNAs including 359 preciously identified miRNAs 23 novel miRNA candidates were obtained. The nucleotide bias analysis of identified miRNAs showed that the miRNAs in Pekin duck was high conserved. The expression of identified miRNAs were significantly different between E13 and E19 as well as between E27 and E19. Fifteen identified miRNAs validated using stem-loop qRT-PCR can be divided into three groups: those with peak expression at E19, those with minimal expression at E19, and those with continuous increase from E11 to E27. Considering that E19 is the fastest growth stage of embryonic Pekin duck breast muscle, these three groups of miRNAs might be the potential promoters, the potential inhibitors, and the potential sustainer for breast muscle growth. Among the 23 novel miRNAs, novel-miRNA-8 and novel-miRNA-14 had maximal expression at some stages. The stem-loop qRT-PCR analysis of the two novel miRNAs and their two targets (MAP2K1 and PPARα) showed that the expression of novel-mir-8 and PPARα reached the lowest points at E19, while that of novel-mir-14 and MAP2K1 peaked at E19, suggesting novel-miRNA-8 and novel-miRNA-14 may be a potential inhibitor and a potential promoter for embryonic breast muscle development of duck. In summary, these results not only provided an overall insight into the miRNAs landscape in embryonic breast muscle of duck, but also a basis for the further investigation of the miRNAs roles in duck skeletal muscle development.

Kelch proteins: emerging roles in skeletal muscle development and diseases.

Our understanding of genes that cause skeletal muscle disease has increased tremendously over the past three decades. Advances in approaches to genetics and genomics have aided in the identification of new pathogenic mechanisms in rare genetic disorders and have opened up new avenues for therapeutic interventions by identification of new molecular pathways in muscle disease. Recent studies have identified mutations of several Kelch proteins in skeletal muscle disorders. The Kelch superfamily is one of the largest evolutionary conserved gene families. The 66 known family members all possess a Kelch-repeat containing domain and are implicated in diverse biological functions. In skeletal muscle development, several Kelch family members regulate the processes of proliferation and/or differentiation resulting in normal functioning of mature muscles. Importantly, many Kelch proteins function as substrate-specific adaptors for Cullin E3 ubiquitin ligase (Cul3), a core component of the ubiquitin-proteasome system to regulate the protein turnover. This review discusses the emerging roles of Kelch proteins in skeletal muscle function and disease.

Myf5 expression during fetal myogenesis defines the developmental progenitors of adult satellite cells

Myf5 is a member of the muscle-specific determination genes and plays a critical role in skeletal muscle development. Whereas the expression of Myf5 during embryonic and fetal myogenesis has been extensively studied, its expression in progenitors that will ultimately give rise to adult satellite cells, the stem cells responsible for muscle repair, is still largely unexplored. To investigate this aspect, we have generated a mouse strain carrying a CreER coding sequence in the Myf5 locus. In this strain, Tamoxifen-inducible Cre activity parallels endogenous Myf5 expression. Combining Myf5CreER and Cre reporter alleles, we were able to evaluate the contribution of cells expressing Myf5 at distinct developmental stages to the pool of satellite cells in adult hindlimb muscles. Although it was possible to trace back the origin of some rare satellite cells to a subpopulation of Myf5+ve progenitors in the limb buds at the late embryonic stage (∼E12), a significant number of satellite cells arise from cells which expressed Myf5 for the first time at the fetal stage (∼E15). These studies provide direct evidence that adult satellite cells derive from progenitors that first express the myogenic determination gene Myf5 during fetal stages of myogenesis.

Stac3 Is a Novel Regulator of Skeletal Muscle Development in Mice

The goal of this study was to identify novel factors that mediate skeletal muscle development or function. We began the study by searching the gene expression databases for genes that have no known functions but are preferentially expressed in skeletal muscle. This search led to the identification of the Src homology three (SH3) domain and cysteine rich (C1) domain 3 (Stac3) gene. We experimentally confirmed that Stac3 mRNA was predominantly expressed in skeletal muscle. We determined if Stac3 plays a role in skeletal muscle development or function by generating Stac3 knockout mice. All Stac3 homozygous mutant mice were found dead at birth, were never seen move, and had a curved body and dropping forelimbs. These mice had marked abnormalities in skeletal muscles throughout the body, including central location of myonuclei, decreased number but increased cross-sectional area of myofibers, decreased number and size of myofibrils, disarrayed myofibrils, and streaming Z-lines. These phenotypes demonstrate that the Stac3 gene plays a critical role in skeletal muscle development and function in mice.

Next-generation sequencing of the porcine skeletal muscle transcriptome for computational prediction of microRNA gene targets.

BackgroundMicroRNA are a class of small RNAs that regulate gene expression by inhibiting translation of protein encoding transcripts through targeting of a microRNA-protein complex by base-pairing of the microRNA sequence to cognate recognition sequences in the 3′ untranslated region (UTR) of the mRNA. Target identification for a given microRNA sequence is generally accomplished by informatics analysis of predicted mRNA sequences present in the genome or in databases of transcript sequence for the tissue of interest. However, gene models for porcine skeletal muscle transcripts in current databases, specifically complete sequence of the 3′ UTR, are inadequate for this exercise.Methodology/Principal FindingsTo provide data necessary to identify gene targets for microRNA in porcine skeletal muscle, normalized cDNA libraries were sequenced using Roche 454 GS-FLX pyrosequencing and de novo assembly of transcripts enriched in the 3′ UTR was performed using the MIRA sequence assembly program. Over 725 million bases of sequence were generated, which assembled into 18,202 contigs. Sequence reads were mapped to a 3′ UTR database containing porcine sequences. The 3′ UTR that mapped to the database were examined to predict targets for previously identified microRNA that had been separately sequenced from the same porcine muscle sample used to generate the cDNA libraries. For genes with microRNA-targeted 3′ UTR, KEGG pathways were computationally determined in order to identify potential functional effects of these microRNA-targeted transcripts.ConclusionsThrough next-generation sequencing of transcripts expressed in skeletal muscle, mapping reads to a 3′ UTR database, and prediction of microRNA target sites in the 3′ UTR, our results identified genes expressed in porcine skeletal muscle and predicted the microRNA that target these genes. Additionally, identification of pathways regulated by these microRNA-targeted genes provides us with a set of genes that can be further evaluated for their potential role in skeletal muscle development and growth.

MicroRNA-27a promotes myoblast proliferation by targeting myostatin

MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs that play critical roles in skeletal muscle development as well as in regulation of muscle cell proliferation and differentiation. However, the role of miRNAs in myoblast proliferation remains poorly understood. Here we found that the expression of miR-27a was increased during proliferation of C2C12 myoblasts. Moreover, overexpression of miR-27a in C2C12 cells promoted myoblast proliferation by reducing the expression of myostatin, a critical inhibitor of skeletal myogenesis. In addition, the miR-27a was confirmed to target myostatin 3′UTR by a luciferase reporter analysis. Together, these results suggest that miR-27a promotes myoblast proliferation through targeting myostatin.