MiR-501-3p Forms a Feedback Loop with FOS, MDFI, and MyoD to Regulate C2C12 Myogenesis

Skeletal muscle plays an essential role in maintaining body energy homeostasis and body flexibility. Loss of muscle mass leads to slower wound healing and recovery from illness, physical disability, poor quality of life, and higher health care costs. So, it is critical for us to understand the mechanism of skeletal muscle myogenic differentiation for maintaining optimal health throughout life. miR-501-3p is a novel muscle-specific miRNA, and its regulation mechanism on myoblast myogenic differentiation is still not clear. We demonstrated that FOS was a direct target gene of miR-501-3p, and MyoD regulated miR-501-3p host gene Clcn5 through bioinformatics prediction. Our previous laboratory experiment found that MDFI overexpression promoted C2C12 myogenic differentiation and MyoD expression. The database also showed there is an FOS binding site in the MDFI promoter region. Therefore, we hypothesize that miR-501-3p formed a feedback loop with FOS, MDFI, and MyoD to regulate myoblast differentiation. To validate our hypothesis, we demonstrated miR-501-3p function in the proliferation and differentiation period of C2C12 cells by transfecting cells with miR-501-3p mimic and inhibitor. Then, we confirmed there is a direct regulatory relationship between miR-501-3p and FOS, MyoD and miR-501-3p, FOS and MDFI through QPCR, dual-luciferase reporter system, and ChIP experiments. Our results not only expand our understanding of the muscle myogenic development mechanism in which miRNA and genes participate in controlling skeletal muscle development, but also provide treatment strategies for skeletal muscle or metabolic-related diseases in the future.