Mdfi Regulates Myoblast Proliferation and Cell Cycle Progression through Cyclins
TL;DR
Mdfi functions as a negative regulator of myoblast proliferation by inducing G1 phase arrest, potentially through modulation of cyclin D within the cyclin-CDK-P21 pathway and is identified as a potential therapeutic target for muscle regeneration and repair.
Abstract
: Background: The inhibitor of MyoD family ( Mdfi ) has been characterized as a myogenic repressor that regulates transcription factor activity through cytoplasmic retention; however, its specific function in myoblast proliferation remains poorly understood. This study aimed to elucidate the precise role of Mdfi in regulating myoblast cell cycle progression and proliferation using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing. Methods: We employed the CRISPR/Cas9 system to construct Mdfi-knockout ( Mdfi − / − ) C2C12 cell lines. Cell cycle distribution was analyzed by flow cytometry, proliferation was assessed by EdU incorporation assays, and molecular mechanisms were investigated through quantitative RT-PCR, Western blotting, and RNA sequencing (RNA-Seq). Differentially expressed genes (DEGs) between wild-type (WT) and Mdfi -overexpressing ( Mdfi -OE) were identified and subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Results: Mdfi overexpression significantly increased the proportion of cells in G1 ( p < 0.001) phase while reducing S and G2 phase populations ( p < 0.001), accompanied by decreased EdU-positive cells. Conversely, Mdfi knockout promoted cell cycle progression into S phase ( p < 0.05) and enhanced proliferation. Mechanistically, Mdfi overexpression downregulated cyclin B1 ( Ccnb1 ) ( p < 0.01), cyclin D1 ( Ccnd1 ) ( p < 0.05), and proliferating cell nuclear antigen ( Pcna ) ( p < 0.05), while upregulating the cyclin-dependent kinase inhibitor P21 ( p < 0.01). RNA-Seq analysis identified 889 DEGs (FDR < 0.05 and |log 2 FC| > 1), with enrichment in cell cycle and calcium signaling pathways. Conclusions: Mdfi functions as a negative regulator of myoblast proliferation by inducing G1 phase arrest, potentially through modulation of cyclin D within the cyclin-CDK-P21 pathway. These findings advance our understanding of the molecular mechanisms governing muscle cell proliferation and identify Mdfi as a potential therapeutic target for muscle regeneration and repair.