5-Aza-dC对牛成肌细胞MyoD1启动子甲基化及mRNA表达的影响

Effects of 5-Aza-dC on MyoD1 Promoter Methylation and mRNA Expression in Bovine Myoblasts

旨在探究5-氮杂-2-脱氧胞苷(5-Aza-2’-deoxycytidine,5-Aza-dC)对牛成肌细胞的增殖和肌分化因子(myogenic differentiation 1,MyoD1)启动子甲基化以及mRNA表达的影响。本研究复苏了前期冻存的关岭牛成肌细胞,并进行培养,待其生长到对数生长期,再通过不同浓度的5-Aza-dC对牛成肌细胞进行处理,利用流式细胞仪检测细胞凋亡和周期;结合高通量检测方法检测MyoD1启动子甲基化水平,并利用qRT-PCR检测MyoD1及相关基因的表达水平。研究发现,0.1μmol·L^(-1) 5-Aza-dC为最适浓度,该浓度下细胞抗凋亡因子Bcl的表达极显著降低(P<0.01),促凋亡因子Bax的表达极显著提高(P<0.01),促凋亡因子Caspase-9的表达显著提高(P<0.05);周期因子Cyclin A2的表达显著提高(P<0.05),而细胞因子Cyclin B1、Cyclin D的表达无显著变化;检测空白组和试验组MyoD1启动子甲基化水平发现,试验组甲基化水平极显著低于空白组(P<0.01);而mRNA的表达水平极显著高于空白组(P<0.01)。5-Aza-dC能够通过改变Caspase-9、Bcl、Bax、Cyclin A2等基因的表达来调节关岭牛成肌细胞的增殖和凋亡,并能够有效降低MyoD1基因启动子的甲基化水平(P<0.01);极显著提高其mRNA的表达量(P<0.01)。低浓度的5-Aza-dC能通过促进细胞凋亡及调控关岭牛MyoD1的甲基化水平来调控MyoD1的表达;同时推测,MyoD1基因启动子的甲基化水平能够影响关岭牛成肌细胞的生长发育,可为遗传标记辅助关岭牛的改良提供理论参考。

The aim of this study was to investigate the effects of 5-Aza-2’-deoxycytidine(5-Aza-dC)on the proliferation of bovine myoblasts,promoter methylation and mRNA expression of myogenic differentiation 1(MyoD1).In this study,the Guanling cattle myoblasts cryopreserved in the early stage were recovered and cultured.After they had grown to the logarithmic growth phase,the bovine myoblasts were treated by different concentrations of 5-Aza-dC.The flow cytometry was used to detect cell apoptosis and cycle.The methylation level of MyoD1 promoter was detected by high-throughput detection methods,the expression level of MyoD1 and related genes were detected by qRT-PCR.The results showed that 0.1μmol·L^(-1) 5-Aza-dC was the optimal concentration,and the expression of anti-apoptotic factor Bcl was significantly decreased(P<0.01),the expression of pro-apoptotic factor Bax was significantly increased(P<0.01),and the expression of pro-apoptotic factor Caspase-9 was significantly increased(P<0.05);The expression of Cyclin A2 was significantly increased(P<0.05),but the expression of Cyclin B1 and Cyclin D were not significantly affected.The MyoD1 promoter methylation level in blank group and experimental group was detected,and the MyoD1 promoter methylation level in experimental group was significantly lower than that in blank group(P<0.01),while the mRNA expression level was significantly higher in experimental group than that in blank group(P<0.01).In summary,5-Aza-dC can regulate the proliferation and apoptosis of Guanling cattle myoblasts by changing the expressions of Caspase-9,Bcl,Bax,Cyclin A2 and other genes,and can effectively reduce the methylation level of MyoD1 gene promoter(P<0.01),and significantly increase its mRNA expression(P<0.01).Low concentration of 5-Aza-dC can promote cell apoptosis and regulate MyoD1 methylation level to regulate the expression of MyoD1 in Guanling cattle.At the same time,it is speculated that the methylation level of the MyoD1 gene promoter can affect the growth and development of Guanling cattle myoblasts,which can provide a theoretical reference for screening genetic markers to assist the improvement of Guanling cattle.