TY  - EJOU
AU  - Jiao, Yiren 
AU  - Cao, Lingbo 
AU  - Xu, Jing 
AU  - Sun, Hongkai 
AU  - Shi, Jia 
AU  - Ma, Xiaoli 
AU  - Wang, Zehao 
AU  - Zhou, Baozhen 

TI  - Mdfi Regulates Myoblast Proliferation and Cell Cycle Progression through Cyclins
T2  - BIOCELL

PY  - 
VL  - 
IS  - 
SN  - 1667-5746

AB  - <b>Background:</b> The inhibitor of MyoD family (<i>Mdfi</i>) 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 <i>Mdfi</i> 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. <b>Methods:</b> We employed the CRISPR/Cas9 system to construct Mdfi-knockout (<i>Mdfi</i><sup>−/−</sup>) 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 <i>Mdfi</i>-overexpressing (<i>Mdfi</i>-OE) were identified and subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. <b>Results:</b> Mdfi overexpression significantly increased the proportion of cells in G1 (<i>p</i> &lt; 0.001) phase while reducing S and G2 phase populations (<i>p</i> &lt; 0.001), accompanied by decreased EdU-positive cells. Conversely, <i>Mdfi</i> knockout promoted cell cycle progression into S phase (<i>p</i> &lt; 0.05) and enhanced proliferation. Mechanistically, Mdfi overexpression downregulated cyclin B1 (<i>Ccnb1</i>) (<i>p</i> &lt; 0.01), cyclin D1 (<i>Ccnd1</i>) (<i>p</i> &lt; 0.05), and proliferating cell nuclear antigen (<i>Pcna</i>) (<i>p</i> &lt; 0.05), while upregulating the cyclin-dependent kinase inhibitor P21 (<i>p</i> &lt; 0.01). RNA-Seq analysis identified 889 DEGs (FDR &lt; 0.05 and |log<sub>2</sub>FC| &gt; 1), with enrichment in cell cycle and calcium signaling pathways. <b>Conclusions:</b> 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.
KW  - Inhibitor of MyoD family (Mdfi); myoblasts; cell cycle arrest; clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9); cyclin; cyclin-dependent kinase inhibitor

DO  - 10.32604/biocell.2026.083738