The miR-6631-5p/ACYP2 Axis Orchestrates Myogenesis and Slow-Twitch Muscle Fiber Specification in Broilers

Poultry skeletal muscle fiber composition, primarily defined by oxidative slow-twitch and glycolytic fast-twitch fibers, is a major determinant of meat quality. Yet the miRNA-mRNA regulatory mechanisms controlling fiber type specification remain largely unresolved. Here, we compared the transcriptomic profiles of pectoralis major (PM) and soleus muscle (SM) in Tianfu broilers and identified gga-miR-6631-5p as a key slow-twitch-associated microRNA (miRNA) through small RNA sequencing and weighted gene co-expression network analysis. Mechanistically, acylphosphatase 2 (ACYP2) was validated as a direct target of gga-miR-6631-5p by bioinformatics prediction and dual-luciferase reporter assays, and its expression was inversely correlated with miR-6631-5p. Functional analyses in primary chicken skeletal muscle satellite cells (SMSCs) revealed that miR-6631-5p significantly promotes cell proliferation and slow-twitch fiber specification but suppresses myogenic differentiation. In contrast, ACYP2 exerts opposite effects by inhibiting SMSCs' proliferation, enhancing myotube fusion, and repressing slow-twitch fiber formation. Finally, rescue assays involving miR-6631-5p and ACYP2 further validated the effect of this regulatory axis on muscle fiber type switching. Together, these results establish that the miR-6631-5p/ACYP2 axis is an important regulator of myogenesis and muscle fiber type specification, highlighting its potential for molecular improvement of poultry meat quality.