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.

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.

Background: There have been many studies on the relationship between circRNAs and fat deposition. Although the liver is a central organ for fat metabolism, there are few reports on the relationship between circRNAs in the liver and fat deposition. Methods: In this study, we systematically analyzed circular RNAs in the liver of Ningxiang pigs, at four time points after birth (30 days, 90 days, 150 days and 210 days). Results: A total of 3705 circRNAs were coexpressed in four time periods were found, and KEGG analysis showed that the significantly upregulated pathways were mainly enriched in lipid metabolism and amino acid metabolism, while significantly downregulated pathways were mainly related to signal transduction, such as ECM–receptor interaction, MAPK signaling pathway, etc. Short time-series expression miner (STEM) analysis showed multiple model spectra that were significantly enriched over time in the liver. By constructing a competing endogenous RNA (ceRNA) regulatory network, 9187 pairs of networks related to the change in development time were screened. Conclusions: The expression profiles of circRNAs in Ningxiang pig liver were revealed at different development periods, and it was determined that there is differential coexpression. Through enrichment analysis of these circRNAs, it was revealed that host genes were involved in metabolism-related signaling pathways and fatty acid anabolism. Through STEM analysis, many circRNAs involved in fat metabolism, transport, and deposition pathways were screened, and the first circRNA–miRNA–mRNA regulation network map in Ningxiang pig liver was constructed. The highly expressed circRNAs related to fat deposition were verified and were consistent with RNA-Seq results.

Growth retardation is a globally prevalent clinical issue, particularly in preterm offspring. It frequently occurs during the early postnatal development of piglets and results in high mortality. In addition to slow postnatal growth caused by complications from immature organs, these offspring are also at risk of facing significant long-term health challenges in adulthood. The liver plays a crucial role in regulating nutrient metabolism and immune function and frequently communicates with other organs to maintain overall health. However, the multifaceted mechanisms by which the liver regulates growth retardation in offspring remain largely unexplored. Here, we selected piglets exhibiting extreme weight variations at both birth and weaning stages for comprehensive histomorphological and multi-omics analysis in the liver. Compared to high weaning weight (HWW) piglets, the liver of low weaning weight (LWW) piglets was predominantly characterized by hepatic vacuolation and structural lesions. Transcriptomic analysis of the liver indicated that differentially expressed genes (DEGs) linked to amino acid metabolism (IDO1, DAO, and UROC1) and innate immune (ISG15, RSAD2, and IFIT1) showed significant upregulation in the high birth weight (HBW) piglets when compared to the low birth weight (LBW) piglets. Furthermore, DEGs identified at the weaning stage are primarily enriched in PPAR signaling pathway, protein digestion and absorption, glutathione metabolism, tryptophan metabolism, and ferroptosis. Notably, ferroptosis was the only pathway that showed overlapping enrichment in the KEGG enrichment analysis across all three omics datasets. Gene GCLM and significantly differential metabolites (SDMs) (L-glutathione, L-cysteine, and Gamma-glutamylcysteine) were co-enriched in ferroptosis and showed higher levels in HWW piglets. The elevation of these genes and metabolites plays a key role in scavenging reactive oxygen species (ROS) accumulation and suppressing hepatic ferroptosis. Notably, GCLM was positively associated with L-glutathione and Gamma-glutamylcysteine, and these two metabolites were positively correlated with weaning weight, implying a potential role of these metabolites in improving growth performance. Finally, integrative analysis of RNA-seq and ATAC-seq data revealed several differentially accessible regions (DARs), which were annotated to the genes ACSL3, ITGA11, COL6A3, CYP2C49, and STXBP3. The transcription factor (TF)-gene regulatory network revealed that five DEGs (PML, STAT1, RCOR2, ELF3, and IRF1) are the downstream target genes of TFs, including MAZ and KIF1. These target DEGs are involved in liver development and the pathogenesis of hepatic disease. Taken together, the genes and metabolites identified in this study provide gene-targeted and nutrition-based therapeutic strategies for piglets with growth limitations during the lactation and even post-weaning stages. Our findings also provide epigenetic insights into the molecular mechanisms underlying liver-mediated modulation of growth and development in offspring.