Methylation Of MEF2A Gene And Its Effect On Myoblasts In Guanling Cattle

Myocyte enhancer factor 2A(MEF2A)plays a critical role in promoting muscle cell growth and differentiation,is widely expressed in various tissues and organs,and has close involvement in many cellular processes including muscle development.Myogenesis is a complex and coordinated process that depends on the mutual regulation of various intracellular and extracellular signals and factors.Epigenetic studies have shown that DNA methylation has a very important impact on muscle generation.DNA methylation has been proved to be a relatively mature and effective epigenetic mode,while promoter DNA methylation generally inhibits gene expression,and its change plays a switch role ingene expression,which can maintain the normal cell function of the body.In this study,the MEF2 A gene expression level and promoter methylation status in tissues of Guanling cattle were analyzed.Secondly,the overexpression and interference plasmids of MEF2 A gene were constructed and successfully transf-ected into primary Guanling cattle myoblasts to analyze the regulatory effect of MEF2 A gene on myoblasts and the influence of MEF2 A expression on the me-thylation level of its promoter.Finally,5-Aza-dC,a methylation inhibitor,was added to primary Guanling cattle myogenic cells to analyze the impact of cha-nges in the methylation status of the MEF2 A promoter on its expression level and myogenic cell differentiation.This further verified the regulatory relati-onship between the methylation status and expression level of MEF2 A.The main research results are as follows:1 Analysis of MEF2 A gene expression and promoter methylation levelThe expression of MEF2 A gene in tissues of Guanling calves and adult cattle was detected by qRT-PCR,and the results of MEF2 A promoter methylation level obtained by bisulfite sequencing(BSP)were analyzed jointly to explore the relationship between MEF2 A gene expression and its promoter methylation level.The qRT-PCR results demonstrated the expression levels of the MEF2 A gene in Guanling calf cattle to be as follows: lung > heart > fat > liver > longissimus dorsi muscle >kidney;In adult Guanling cattle,it is heart > lung > liver > longissimus dorsi muscle >fat > kidney.BSP results showed that the levels of MEF2 A promoter methylation in the heart,liver,lungs,kidneys,longissimus dorsi muscle,and fat of Guanling calves were 2.08%,2.28%,1.39%,1.39%,1.42%,and 1.49%,respectively.At the same time,it was detected that the methylation levels of the MEF2 A gene promoter region in the heart and fat of adult Guanling cattle were 2.68% and 2.21%,respectively,which were significantly higher than the methylation levels in the same tissue of calves(P<0.01),while the expression levels in each tissue of adult cattle were significantly lower than those of calves(P<0.01).The results showed that there was a significant negative correlation between the expression of MEF2 A gene and its promoter methylation level.2 Effect of MEF2 A expression level on myoblastsThe pEGFP-C1-MEF2 A overexpression plasmid was successfully constructed,and the sh RNA-MEF2A-3 interference plasmid with the best efficiency was synthesized and detected.The overexpression plasmid and interference plasmid of MEF2 A were transfected into the primary myoblasts of Guanling cattle,respectively.The effects of MEF2 A expression on the proliferation,cell cycle,apoptosis,and secretion of growth hormone(GH)and insulin(INS)were evaluated by CCK8 assay,cell cycle and apoptosis analysis,and ELISA.The results of CCK8 showed that overexpression of MEF2 A significantly upregulated myoblast proliferation activity(P<0.01),while inhibition of MEF2 A had the opposite effect.Cell cycle results showed that overexpression of MEF2 A could accelerate the progression of a large number of myoblasts from G1 to S phase,and inhibition of MEF2 A would block the progression from G1 to S phase.Further testing of growth hormone(GH)and insulin(INS)levels revealed that overexpression of MEF2 A gene significantly promoted GH production(P<0.01),but had no significant effect on INS production(P>0.05);Inhibiting the MEF2 A gene significantly reduced the levels of both GH and INS(P<0.01).The changes in the expression of Myo D1,IGF1,CDK2,andCCNA2 genes after qRT-PCR detection of overexpression and inhibition of MEF2 A were consistent with the above results.In addition,it was found that MEF2 A gene had a certain role in resisting apoptosis.Overexpression of MEF2 A significantly upregulated BCL2 and downregulated BAD gene expression(P<0.01),while inhibition of MEF2 A significantly downregulated BCL2 expression(P<0.01),but had no effect on BAD(P>0.05).3 Effect of MEF2 A expression level on promoter methylation levelAfter MEF2 A overexpression and interference plasmids were transfected into primary myoblasts of Guanling cattle,the changes in DNMT1 gene expression in myoblasts were detected by qRT-PCR,and the methylation rate of MEF2 A promoter in the overexpression and interference groups was detected by BSP method.The results of qRT-PCR showed that after overexpression of MEF2 A,the expression of DNMT1 was significantly decreased(P<0.01),while inhibition of MEF2 A upregulated the expression of DNMT1,with a significant difference(P<0.01).BSP detection showed that the methylation level of MEF2 A promoter in the overexpression group decreased from 3.00% to 2.41%,while the methylation level of MEF2 A promoter in the interference group increased from 2.05% to 2.31%.The results showed that both high and low expression levels of MEF2 A gene can lead to changes in the methylation rate of its promoter.4 Effects of 5-Aza-dC on MEF2 A promoter methylation and Guanling bovine myoblastsBased on previous studies,5-Aza-dC was added to Guanling cattle myoblasts with 0.1 μmol/L,1 μmol/L and 5 μmol/L,respectively,to explore the effects of5-Aza-dC on the methylation rate of MEF2 A gene promoter region and the proliferation and development of Guanling cattle myoblasts.Compared with blank group,methylation rate of MEF2 A promoter all decreased at different concentrations of 5-Aza-dC,with the 0.1 μmol/L group showing a significant difference(P<0.05).qRT-PCR results also showed that the expression levels of DNMT1 in all treatment groups were significantly downregulated(P<0.01),with the lowest expression level observed in the 0.1 μmol/L group,corresponding to the methylation rate results.Furthermore,both the 0.1 μmol/L and 1 μmol/L groups significantly upregulated MEF2 A gene expression(P<0.01),with the highest expression level observed in the1 μmol/L group,while the 5 μmol/L group showed no significant difference(P>0.05).These findings once again confirm the negative correlation between MEF2 A gene expression and its promoter methylation level.According to the results,the CCK8 assay showed that cell viability in all drug-treated groups was significantly lower than that in the control group(P<0.01),indicating the cytotoxicity and differentiation inducing effects of 5-Aza-dC.The apoptosis experiment also showed that all the dosed groups could promote apoptosis,and the expression levels of BAD and BCL2 were also corresponding to the apoptosis results.Further detection using cell cycle analysis and ELISA showed that the 1 μmol/L group could shorten the cell cycle process significantly(P<0.05),and also significantly increase the content of GH and INS in differentiated myocytes(P<0.01).qRT-PCR results also showed that MyoD1 expression could be upregulated in 1 μmol/L group(P<0.05),and the expression levels of IGF1,CDK2 andCCNA2 genes were significantly increased,indicating that1 μmol/L 5-Aza-dC could promote the proliferation of Guanling cattle myoblasts.The above results demonstrated that MEF2 A gene could promote the proliferation of Guanling cattle myoblasts.MEF2 A gene expression was negatively correlated with methylation rate at both tissue and cell levels.1 μmol/L concentration of 5-Aza-dC can reduce MEF2 A methylation rate,increase MEF2 A gene expression and promote myoblast proliferation.