Asparagine synthetase regulates the proliferation and differentiation of chicken skeletal muscle satellite cells

Objective: Asparagine synthetase (ASNS) is an aminotransferase responsible for the bio synthesis of aspartate by using aspartic acid and glutamine. ASNS is highly expressed in fastgrowing broilers, but few studies have reported the regulatory role of ASNS in muscle development. Methods: To explore the function of ASNS in chicken muscle development, the expression of ASNS in different chicken breeds and tissues were first performed by realtime quantitative reverse transcription polymerase chain reaction (RTPCR). Then, using realtime quantitative RTPCR, western blot, EdU assay, cell cycle assay and immunofluorescence, the effects of ASNS on the proliferation and differentiation of chicken skeletal muscle satellite cell (SMSC) were investigated. Finally, potential mechanisms by which ASNS influences chicken muscle fiber differentiation were identified through RNASeq. Results: The mRNA expression pattern of ASNS in muscles mirrors trends in muscle fiber crosssectional area, average daily weight gain, and muscle weight across different breeds. ASNS knockdown inhibited SMSC proliferation, while overexpression showed the opposite. Moreover, ASNS attenuated SMSC differentiation by activating the adenosine 5'mono phosphate (AMP) activated protein kinase (AMPK) pathway. Additionally, 5aminoimidazole 4carboxamide1 beta Dribofuranoside (AICAR) treatment suppressed the cell differentiation induced by siRNAASNS. RNASeq identified 1,968 differentially expressed genes (DEGs) during chicken SMSC differentiation when overexpression ASNS. Gene ontology (GO) enrichment analysis revealed that these DEGs primarily participated in 8 biological processes, 8 cellular components, and 4 molecular functions. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis identified several significantly enriched signaling pathways, such as the JAKSTAT signaling pathway, tumor necrosis factor signaling pathway, toll like receptor signaling pathway, and PI3KAkt signaling pathway. Conclusion: ASNS promotes proliferation while inhibits the differentiation of chicken SMSCs. This study provides a theoretical basis for studying the role of ASNS in muscle development.