This study investigates the regulatory effects of methylation in the promoter region of the bovine MEF2A gene on its transcription levels and the impact on bovine myoblasts. Transcription levels and promoter methylation status of MEF2A in the same tissues of calves and adult cattle were assessed using qRT-PCR and BSP methods. The results indicated that MEF2A expression levels in calves were significantly lower than those in adult cattle (P <
0.05), while the methylation rate of MEF2A was significantly higher in calves (P <
0.05), suggesting a correlation between high methylation levels and reduced gene expression. Subsequently, MEF2A overexpression and interference vectors were transfected into bovine myoblasts to examine the effects of altered MEF2A expression on its promoter methylation status. The findings revealed that MEF2A overexpression significantly reduced the methylation rate (P <
0.01), whereas MEF2A interference increased the methylation rate (P <
0.01), aligning with the expression trends of DNMT1. Furthermore, bovine myoblasts were treated with varying concentrations of the methylation inhibitor 5-Aza-dC to evaluate changes in MEF2A promoter methylation and mRNA levels. The effects on cell cycle progression, apoptosis, and other growth parameters were assessed using flow cytometry, ELISA, and qRT-PCR. Results showed that a concentration of 1 μM 5-Aza-dC effectively reduced MEF2A promoter methylation and significantly upregulated MEF2A expression, leading to accelerated cell cycle progression and increased secretion levels of GH and INS, all differences being statistically significant (P <
0.01). Additionally, 1 μM of 5-Aza-dC promoted apoptosis, with qRT-PCR results for relevant genes supporting this finding. In conclusion, methylation of the MEF2A promoter negatively regulates its mRNA transcription levels, thereby impacting the growth and development of Guanling cattle myoblasts. These results provide valuable insights for the genetic improvement of cattle through marker-assisted selection.