Mdfi Regulates Myoblast Proliferation and Cell Cycle Progression through Cyclins
Yiren Jiao1,*, Lingbo Cao2, Jing Xu1, Hongkai Sun1, Jia Shi3, Xiaoli Ma1, Zehao Wang1,*, Baozhen Zhou1,*
1 College of Medicine, Xi’an International University, Xi’an, China
2 School of Geography and Tourism, Jiaying University, Meizhou, China
3 National Engineering Research Center for Breeding Swine Industry, Guangdong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, China
* Corresponding Author: Yiren Jiao. Email: 
; Zehao Wang. Email: 
; Baozhen Zhou. Email: 
BIOCELL https://doi.org/10.32604/biocell.2026.083738
Received 09 April 2026; Accepted 24 July 2026; Published online 06 August 2026
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
2FC| > 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.
Keywords
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
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