The molecular mechanism by which miR-206-regulated JunD expression influences myoblast proliferation and differentiation in yaks.

Jun dimerization protein D (JunD), a member of the activating protein-1 transcription factor family, serves as a key regulator of mammalian myogenesis by orchestrating cell cycle progression and coordinating the network of myogenic differentiation determinants. miR-206 exhibits tissue-specific expression in skeletal muscle, with abundant representation across miRNA expression profiles in multiple mammalian species. Although both JunD and miR-206 are critically involved in muscle development, their specific roles in yak skeletal muscle ontogeny remain poorly characterized, particularly regarding the regulatory axis involving miR-206-mediated targeting of JunD during myoblast proliferation and differentiation. To address this knowledge gap, this study used methods such as CCK-8, EdU, RT-qPCR, western blot, immunofluorescence, and the dual-luciferase reporter system. It was found that JunD significantly promoted the expression of cell cycle factors, such as CDK2 and PCNA, and increased cell proliferation and the proportion of S-phase cells. JunD overexpression or interference experiments demonstrated that it enhanced the differentiation and myotube formation ability of myoblasts and simultaneously upregulated the expression of key genes such as MYOG and MYOD. Additionally, the results of cell cycle detection by flow cytometry revealed that the JunD gene inhibited the apoptosis of yak myoblasts. Transcriptome analysis revealed that JunD regulated cAMP and other signaling pathways related to proliferation, differentiation, and apoptosis. The results of the dual-luciferase reporter assay showed a good binding relationship between miR-206 and JunD. The rescue experiments demonstrated that miR-206 regulated the expression of the JunD gene, thereby exerting its influence at the transcriptional level. This study marks the first identification of JunD in yaks and clarifies its role in the development of yak myoblasts through the miR-206-JunD regulatory axis. These findings provide new insights into the molecular breeding of cattle, contributing to the basic research into the breeding and muscle development of yaks.