Abstract

Coordinated development and homeostasis among bone, muscle, and adipose tissue are essential for the sustainable advancement of the broiler industry. Bone marrow mesenchymal stem cells (BMSCs) possess multi-lineage differentiation potential and provide an in vitro model for investigating regulatory mechanisms of the “bone-muscle-adipose” balance. Nevertheless, microRNA (miRNA)–mRNA regulatory network and the functions of key miRNAs during myogenic differentiation of chicken BMSCs remain unclear. We profiled mRNA and miRNA expression during this process to identify key miRNAs regulating BMSC proliferation and differentiation. mRNA sequencing (mRNA-seq) and miRNA sequencing (miRNA-seq) before and after 5-azacytidine (5-Aza) induction identified 2,233 differentially expressed genes (DEGs) and 67 differentially expressed miRNAs (DE miRNAs). DEGs were mainly enriched in cell cycle and DNA replication pathways, whereas predicted targets of DE miRNAs were predominantly enriched in calcium signaling pathway. A candidate miRNA–mRNA regulatory network was constructed using 13 skeletal muscle-related DE miRNAs and inversely expressed target mRNAs, with target genes enriched in protein processing in the endoplasmic reticulum and Wnt signaling pathways. RNAhybrid prediction and dual-luciferase reporter assays confirmed that miR-26a-5p directly targets MyoD Family Inhibitor Domain Containing (MDFIC). Functionally, miR-26a-5p overexpression suppressed proliferation markers ( CCNB2, CCND2, CDK8 ) and myogenic markers ( MYOD1, MYOG, MEF2C, MYHC, Desmin ), and reduced MYOD1 protein, whereas miR-26a-5p inhibition produced opposite effects. Collectively, this study delineates the miRNA–mRNA regulatory network during myogenic differentiation of chicken BMSCs and demonstrates that miR-26a-5p negatively regulates proliferation and myogenic differentiation by targeting MDFIC . These findings identify candidate targets for understanding the post-transcriptional regulation of myogenesis in