Retinoic acid signalling inhibits myogenesis by blocking MYOD translation in pig skeletal muscle cells.

Vitamin A is an essential nutrient in animals, playing important roles in animal health. In the pig industry, proper supplementation of vitamin A in the feed can improve pork production performance, while deficiency or excessive intake can lead to growth retardation or disease. However, the specific molecular mechanisms through which vitamin A operates on pig skeletal muscle growth as well as muscle stem cell function remain unexplored. Therefore, in this study, we isolated the pig primary skeletal muscle stem cells (pMuSCs) and treated with retinoic acid (RA), the natural metabolite of vitamin A, and then examined the myogenic capacity of pMuSCs via immunostaining, real-time PCR, CCK8 and western-blot analysis. Unexpectedly, the RA caused a significant decrease in the proliferation and differentiation of pMuSCs. Mechanistically, the RA addition induced the activation of retinoic acid receptor gamma (RARγ), which inhibited the myogenesis through the blockage of protein translation of the master myogenic regulator myogenic differentiation 1 gene (MYOD). Specifically, RARγ inactivate AKT kinase (AKT) signalling and lead to dephosphorylation of eukaryotic translation initiation factor 4E binding protein 1 (eIF4EBP1), which in turn repress the eukaryotic translation initiation factor 4E (eIF4E) complex and block mRNA translation of MYOD. Inhibition of AKT could rescue the myogenic defects of RA-treated pMuSCs. Our findings revealed that retinoid acid signalling inhibits the skeletal muscle stem cell proliferation and differentiation in pigs. Therefore, the vitamin A supplement in the feedstuff should be cautiously optimized to avoid the potential adverse consequences on muscle development associated with the excessive levels of retinoic acid.

Retinoic acid and RARγ maintain satellite cell quiescence through regulation of translation initiation.

In adult skeletal muscle, satellite cells are in a quiescent state, which is essential for the future activation of muscle homeostasis and regeneration. Multiple studies have investigated satellite cell proliferation and differentiation, but the molecular mechanisms that safeguard the quiescence of satellite cells remain largely unknown. In this study, we purposely activated dormant satellite cells by using various stimuli and captured the in vivo-preserved features from quiescence to activation transitions. We found that retinoic acid signaling was required for quiescence maintenance. Mechanistically, retinoic acid receptor gamma (RARγ) binds to and stimulates genes responsible for Akt dephosphorylation and subsequently inhibits overall protein translation initiation in satellite cells. Furthermore, the alleviation of retinoic acid signaling released the satellite cells from quiescence, but this restraint was lost in aged cells. Retinoic acid also preserves the quiescent state during satellite cell isolation, overcoming the cellular stress caused by the isolation process. We conclude that active retinoic acid signaling contributes to the maintenance of the quiescent state of satellite cells through regulation of the protein translation initiation process.

Disturbance of calcium homeostasis and myogenesis caused by TET2 deletion in muscle stem cells.

Skeletal muscle myogenesis is a sophisticated process controlled by genetic and epigenetic regulators. In animals, one of the key enzymes for the DNA demethylation of 5-methylcytosine is TET2. Although TET2 is essential for muscle development, the mechanisms by which TET2 regulates myogenesis, particularly the implication for muscle stem cells, remains unclear. In the present study, we employed the TET2 knockout mouse model to investigate the function of TET2 in muscle development and regeneration. We observed that TET2 deficiency caused impaired muscle stem cell proliferation and differentiation, resulting in the reduction in both myofiber number and muscle tissue size. Specifically, TET2 maintains calcium homeostasis in muscle stem cells by controlling the DNA methylation levels of the calcium pathway genes. Forced expression of the sodium/calcium exchanger protein SLC8A3 could rescue the myogenic defects in TET2 knockout cells. Our data not only illustrated the vital function of TET2 during myogenesis but also identified novel targets that contribute to calcium homeostasis for enhancing muscle function.

MicroRNA miR-509-3p inhibit metastasis and epithelial-mesenchymal transition in hepatocellular carcinoma.

Our study seeks to obtain data which help to assess the impacts and related mechanisms of microRNA miR-509-3p in hepatocellular carcinoma (HCC). We found that the expression of miR-509-3p was down-regulated and Twist was up-regulated in HCC tissues and cell lines (HepG2, HCCLM3, Bel7402, and SMMC7721) compared with the adjacent normal tissues and normal human hepatocyte (L02). Moreover, cell proliferation, invasion, migration and epithelial-mesenchymal transition (EMT) in HepG2 and HCCLM3 cells were appeared to be markedly suppressed by overexpressed miR-509-3p. Overexpression of miR-509-3p also performed inhibition of the growth and metastasis in vivo. In addition, miR-509-3p could target and inhibit Twist expression, and it could further reverse the tumor promotion by Twist in HCC. All in all, miR-509-3p overexpression causes inhibition of the proliferation, migration, invasion and EMT of HCC cells by negatively regulating Twist, thereby suppressing HCC development and metastasis.

Long Non-coding RNA H19 Regulates Porcine Satellite Cell Differentiation Through miR-140-5p/SOX4 and DBN1.

The H19 gene promotes skeletal muscle differentiation in mice, but the regulatory models and mechanisms of myogenesis regulated by H19 are largely unknown in pigs. Therefore, the regulatory modes of H19 in the differentiation of porcine skeletal muscle satellite cells (PSCs) need to be determined. We observed that H19 gene silencing could decrease the expressions of the myogenin (MYOG) gene, myogenic differentiation (MYOD), and myosin heavy chain (MYHC) in PSCs. Therefore, we constructed and sequenced 12 cDNA libraries of PSCs after knockdown of H19 at two differentiation time points to analyze the transcriptome differences. A total of 11,419 differentially expressed genes (DEGs) were identified. Among these DEGs, we found through bioinformatics analysis and protein interaction experiment that SRY-box transcription factor 4 (SOX4) and Drebrin 1 (DBN1) were the key genes in H19-regulated PSC differentiation. Functional analysis shows that SOX4 and DBN1 promote PSC differentiation. Mechanistically, H19 regulates PSC differentiation through two different pathways. On the one hand, H19 functions as a molecular sponge of miR-140-5p, which inhibits the differentiation of PSCs, thereby modulating the derepression of SOX4. On the other hand, H19 regulates PSC differentiation through directly binding with DBN1. Furthermore, MYOD binds to the promoters of H19 and DBN1. The knockdown of MYOD inhibits the expression of H19 and DBN1. We determined the function of H19 and provided a molecular model to elucidate H19's role in regulating PSC differentiation.

Long Noncoding Ribonucleic Acid MSTRG.59589 Promotes Porcine Skeletal Muscle Satellite Cells Differentiation by Enhancing the Function of PALLD.

Skeletal muscle satellite cells are a class of undifferentiated mononuclear myogenic stem cells distributed between the myofibroblast and membrane basement. Since their development determines the development of skeletal muscles, knowledge of their proliferation, differentiation, and fate is vital for understanding skeletal muscle development. Increasing evidence have shown that long noncoding RNA (lncRNA) plays an important role in regulating the development process of satellite cells. Based on the results of our previous studies, we screened lncRNA MSTRG.59589, which is highly expressed in skeletal muscle tissue. In the present study, knockdown of MSTRG.59589 significantly inhibited satellite cell differentiation at various time points, whereas overexpression of MSTRG.59589 demonstrated opposite effects. An MSTRG.59589 knockdown cell model was constructed for transcriptome sequencing, and RNA sequencing analysis screened out a large number of differentially expressed genes. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses of these differentially expressed genes revealed that they are mainly enriched in actin cytoskeleton, muscle contraction, and other pathways related to muscle development. Mechanistic analyses showed that MSTRG.59589 could promote the differentiation process of skeletal muscle satellite cells by positively regulating the expression level of the target gene PALLD. This experiment lays a theoretical foundation for deeper studies on the mechanism of MSTRG.59589 in the differentiation of porcine skeletal muscle satellite cells.

Transcriptome analysis reveals long intergenic non-coding RNAs involved in skeletal muscle growth and development in pig.

Long intergenic non-coding RNAs (lincRNAs) play essential roles in numerous biological processes and are widely studied. The skeletal muscle is an important tissue that plays an essential role in individual movement ability. However, lincRNAs in pig skeletal muscles are largely undiscovered and their biological functions remain elusive. In this study, we assembled transcriptomes using RNA-seq data published in previous studies of our laboratory group and identified 323 lincRNAs in porcine leg muscle. We found that these lincRNAs have shorter transcript length, fewer exons and lower expression level than protein-coding genes. Gene ontology and pathway analyses indicated that many potential target genes (PTGs) of lincRNAs were involved in skeletal-muscle-related processes, such as muscle contraction and muscle system process. Combined our previous studies, we found a potential regulatory mechanism in which the promoter methylation of lincRNAs can negatively regulate lincRNA expression and then positively regulate PTG expression, which can finally result in abnormal phenotypes of cloned piglets through a certain unknown pathway. This work detailed a number of lincRNAs and their target genes involved in skeletal muscle growth and development and can facilitate future studies on their roles in skeletal muscle growth and development.