MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play crucial regulatory roles in many biological processes, including the growth and development of skeletal muscle. miRNA-100-5p is often associated with tumor cell proliferation and migration. This study aimed to uncover the regulatory mechanism of miRNA-100-5p in myogenesis. In our study, we found that the miRNA-100-5p expression level was significantly higher in muscle tissue than in other tissues in pigs. Functionally, this study shows that miR-100-5p overexpression significantly promotes the proliferation and inhibits the differentiation of C2C12 myoblasts, whereas miR-100-5p inhibition results in the opposite effects. Bioinformatic analysis predicted that Trib2 has potential binding sites for miR-100-5p at the 3′UTR region. A dual-luciferase assay, qRT-qPCR, and Western blot confirmed that Trib2 is a target gene of miR-100-5p. We further explored the function of Trib2 in myogenesis and found that Trib2 knockdown markedly facilitated proliferation but suppressed the differentiation of C2C12 myoblasts, which is contrary to the effects of miR-100-5p. In addition, co-transfection experiments demonstrated that Trib2 knockdown could attenuate the effects of miR-100-5p inhibition on C2C12 myoblasts differentiation. In terms of the molecular mechanism, miR-100-5p suppressed C2C12 myoblasts differentiation by inactivating the mTOR/S6K signaling pathway. Taken together, our study results indicate that miR-100-5p regulates skeletal muscle myogenesis through the Trib2/mTOR/S6K signaling pathway.

The growth and development of the Longissimus Dorsi muscle are complex, playing an important role in the determination of pork quality. The study of the Longissimus Dorsi muscle at the mRNA level is particularly crucial for finding molecular approaches to improving meat quality in pig breeding. The current study utilized transcriptome technology to explore the regulatory mechanisms of muscle growth and intramuscular fat (IMF) deposition in the Longissimus Dorsi muscle at three core developmental stages (natal stage on day 1, growing stage on day 60, and finishing stage on day 210) in Ningxiang pigs. Our results revealed 441 differentially expressed genes (DEGs) in common for day 1 vs. day 60 and day 60 vs. day 210, and GO (Gene Ontology) analysis showed that candidate genes RIPOR2, MEGF10, KLHL40, PLEC, TBX3, FBP2, and HOMER1 may be closely related to muscle growth and development, while KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis showed that DEGs (UBC, SLC27A5, RXRG, PRKCQ, PRKAG2, PPARGC1A, PLIN5, PLIN4, IRS2, and CPT1B) involved the PPAR (Peroxisome Proliferator-Activated Receptor) signaling pathway and adipocytokine signaling pathway, which might play a pivotal role in the regulation of IMF deposition. PPI (Protein-Protein Interaction Networks) analysis found that the STAT1 gene was the top hub gene. Taken together, our results provide evidence for the molecular mechanisms of growth and development and IMF deposition in Longissimus Dorsi muscle to optimize carcass mass.

Muscle cell growth plays an important role in skeletal muscle development. Circular RNAs (circRNAs) have been proven to be involved in the regulation of skeletal muscle growth and development. In this study, we explored the effect of circTTN on myoblast growth and its possible molecular mechanism. Using C2C12 cells as a functional model, the authenticity of circTTN was confirmed by RNase R digestion and Sanger sequencing. Previous functional studies have showed that the overexpression of circTTN inhibits myoblast proliferation and differentiation. Mechanistically, circTTN recruits the PURB protein on the Titin (TTN) promoter to inhibit the expression of the TTN gene. Moreover, PURB inhibits myoblast proliferation and differentiation, which is consistent with circTTN function. In summary, our results indicate that circTTN inhibits the transcription and myogenesis of the host gene TTN by recruiting PURB proteins to form heterotypic complexes. This work may act as a reference for further research on the role of circRNA in skeletal muscle growth and development.

The processes of muscle growth and development, including myoblast proliferation, migration, differentiation, and fusion, are modified by a variety of regulatory factors. MYL4 plays an important role in atrial development, atrial cardiomyopathy, muscle-fiber size, and muscle development. The structural variation (SV) of MYL4 was found via the de novo sequencing of Ningxiang pigs, and the existence of SV was verified in the experiments. The genotype distribution of Ningxiang pigs and Large White pigs was detected, and it was found that Ningxiang pigs were mainly of the BB genotype and that Large White pigs were mainly of the AB genotype. However, the molecular mechanisms behind the MYL4-mediated regulation of skeletal muscle development need to be deeply explored. Therefore, RT-qPCR, 3′RACE, CCK8, EdU, Western blot, immunofluorescence, flow cytometry, and bioinformation analysis were used to explore the function of MYL4 in myoblast development. The cDNA of MYL4 was successfully cloned from Ningxiang pigs, and its physicochemical properties were predicted. The expression profiles in six tissues and four stages of Ningxiang pigs and Large White pigs were found to be the highest in the lungs and 30 days after birth. The expression of MYL4 increased gradually with the extension of the myogenic differentiation time. The myoblast function test showed that the overexpression of MYL4 inhibited proliferation and promoted apoptosis and differentiation. The knockdown of MYL4 showed the opposite result. These results enhance our understanding of the molecular mechanisms of muscle development and provide a solid theoretical foundation for further exploring the role of the MYL4 gene in muscle development.

MicroRNAs (miRNAs) are key regulators involved in the myogenic process in skeletal muscles. miR-708-5p plays an important role in various biochemical and physiological processes, but its function in skeletal myogenesis remain unclear. In this study, we first explored the effects of miR-708-5p on C2C12 proliferation and differentiation by overexpression and interference experiments. Then, we predicted the target genes of miR-708-5p and analyzed their function. We found that miR-708-5p was gradually increased during myoblast differentiation. Overexpression of miR-708-5p significantly inhibited cell proliferation and promoted the differentiation of myoblasts. A total of 253 target genes were predicted using a bioinformatics approach. These genes were significantly enriched in muscle growth-related GO terms and KEGG pathways, such as actin filament organization, actin cytoskeleton organization, PI3K-Akt pathway, insulin pathway, and Jak-STAT pathway. Among them, Pik3ca, Pik3r3, and Irs1 were considered to be the key target genes of miR-708-5p. To sum up, miR-708-5p inhibited C2C12 cells proliferation and promoted C2C12 cells differentiation. Its target genes significantly enriched in GO terms and KEGG pathways related to the development and growth of muscle. Our results provided a basis for studies on the function and mechanism of miR-708-5p regulating skeletal muscle growth and development.

With the continuous improvement in living standards, people’s demand for high-quality meat is increasing. Ningxiang pig has delicious meat of high nutritional value, and is loved by consumers. However, its slow growth and low meat yield seriously restrict its efficient utilization. Gene expression is the internal driving force of life activities, so in order to fundamentally improve its growth rate, it is key to explore the molecular mechanism of skeletal muscle development in Ningxiang pigs. In this paper, Ningxiang boars were selected in four growth stages (30 days: weaning period, 90 days: nursing period, 150 days: early fattening period, and 210 days: late fattening period), and the longissimus dorsi (LD) muscle was taken from three boars in each stage. The fatty acid content, amino acid content, muscle fiber diameter density and type of LD were detected by gas chromatography, acidolysis, hematoxylin eosin (HE) staining and immunofluorescence (IF) staining. After transcription sequencing, weighted gene co-expression network analysis (WGCNA) combined with the phenotype of the LD was used to explore the key genes and signaling pathways affecting muscle development. The results showed that 10 modules were identified by WGCNA, including 5 modules related to muscle development stage, module characteristics of muscle fiber density, 5 modules characteristic of muscle fiber diameter, and a module characteristic of palmitoleic acid (C16:1) and linoleic acid (C18:2n6C). Gene ontology (GO) enrichment analysis found that 52 transcripts relating to muscle development were enriched in these modules, including 44 known genes and 8 novel genes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that these genes were enriched in the auxin, estrogen and cyclic guanosine monophosphate-protein kinase G (cGMP-PKG) pathways. Twelve of these genes were transcription factors, there were interactions among 20 genes, and the interactions among 11 proteins in human, pig and mouse were stable. To sum up, through the integrated analysis of phenotype and transcriptome, this paper analyzed the key genes and possible regulatory networks of skeletal muscle development in Ningxiang pigs at various stages, to provide a reference for the in-depth study of skeletal muscle development.