Lianjie Hou

Skeletal muscle plays a crucial role in physical activity and in regulating body energy and protein balance. Myoblast proliferation, differentiation, and apoptosis are indispensable processes for myoblast myogenesis. Profilin 2a (PFN2a) is a ubiquitous actin monomer-binding protein and promotes lung cancer growth and metastasis through suppressing the nuclear localization of histone deacetylase 1 (HDAC1). However, how PFN2a regulates myoblast myogenic development is still not clear. We constructed a C2C12 mouse myoblast cell line overexpressing PFN2a. The CRISPR/Cas9 system was used to study the function of PFN2a in C2C12 myogenic development. We find that PFN2a suppresses proliferation and promotes apoptosis and consequentially downregulates C2C12 myogenic development. The suppression of PFN2a also decreases the amount of HDAC1 in the nucleus and increases the protein level of p53 during C2C12 myogenic development. Therefore, we propose that PFN2a suppresses C2C12 myogenic development via the p53 pathway. Si-p53 (siRNA-p53) reverses the PFN2a inhibitory effect on C2C12 proliferation and the PFN2a promotion effect on C2C12 apoptosis, and then attenuates the suppression of PFN2a on myogenic differentiation. Our results expand understanding of PFN2a regulatory mechanisms in myogenic development and suggest potential therapeutic targets for muscle atrophy-related diseases.

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.