Abstract
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.
Highlights
Myogenesis in the adult pig is a highly regulated process that includes the activation of muscle stem cells, proliferation and differentiation of myoblasts, and cell fusion to form multinucleated myotubes, which eventually differentiate into muscle fiber (Li et al, 2016, 2019; Zhu et al, 2017)
H19 had a higher expression level in the differentiation period than in the proliferation period, and its expression level increased with differentiation time points (Figure 1B), thereby suggesting the possible role of H19 in porcine skeletal muscle satellite cells (PSCs) differentiation
The conservation of H19 was analyzed in human, mouse, and pig (Supplementary Figure S2), and it showed that H19 was partially conservative
Summary
Myogenesis in the adult pig is a highly regulated process that includes the activation of muscle stem cells, proliferation and differentiation of myoblasts, and cell fusion to form multinucleated myotubes, which eventually differentiate into muscle fiber (Li et al, 2016, 2019; Zhu et al, 2017). LncRNA AK017368 can promote proliferation and restrain differentiation of C2C12 cells (Liang et al, 2018). Xu et al (2017) demonstrated that H19 could promote the differentiation of bovine skeletal muscle SCs by suppressing Sirt1/FoxO1. These discoveries demonstrate that H19 plays an important role in porcine skeletal muscle SC (PSC) differentiation and myogenesis through different molecular mechanisms in different species or different tissues. We aimed to explore new molecular mechanisms in this process
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