心肌梗死小鼠心肌组织的m6A甲基化分析

Differential expression and implication of m6A methylation in mice with experimental myocardial infarction

目的发现心肌梗死(MI)小鼠心肌组织中N6-腺苷酸甲基化(m6A)差异修饰的基因并探讨其在MI中可能参与的病理过程。方法取8~10周龄的SPF级C57BL/6J雄性小鼠18只,按照随机数字表法将其分为MI组(n=9)和对照组(n=9)。应用RNA甲基化测序(MeRIP-seq)检测2组小鼠心肌组织的m6A修饰基因,通过生物信息学分析如主成分分析和GO富集分析探索MI小鼠心肌组织RNA甲基化修饰的特征并进行表达量、m6A修饰水平的验证及其功能预测。结果聚类热图分析显示相比于对照组,MI组m6A修饰水平上调和下调的基因分别有537个和364个。主成分分析显示对照组和MI组的m6A修饰差异可显著区分。m6A修饰的特征性序列为GGACU且主要集中在蛋白质编码区。RNA-seq和MeRIP-seq联合分析显示发生差异m6A修饰同时伴随mRNA差异表达的基因有119个。韦恩图显示不同基因m6A修饰差异和mRNA表达差异。GO富集分析显示MI组中发生m6A差异修饰的基因主要参与心脏发育等过程。qPCR验证得知Gbp6水平上升、Dnaja1和Dnajb1水平下降,MeRIP-qPCR验证可知Hspa1b的m6A修饰水平下调。结论MI小鼠心肌组织中存在m6A差异修饰,发生m6A差异修饰的基因可能受甲基化相关酶的影响,进而通过调控凋亡和炎症影响心肌梗死的发生发展。

ObjectiveTo define differentially expressed N6-adenylate methylation(m6A)genes in the myocardial tissue of mice with myocardial infarction(MI)and explore its potential impact on the pathological process of MI.MethodsThe random number table method was used to divide the eighteen SPF C57BL/6J male mice aged from 8 to 10 weeks into MI group(MI group,n=9)and control group(control group,n=9).Modified m6A genes from the myocardial tissue were detected via methylated RNA immunoprecipitation with the next generation sequencing(MeRIP-seq).We explored methylation modified characteristics,verified mRNA expression and m6A modified level by bioinformatics analysis,qPCR and MeRIP-qPCR.ResultsThe Heatmap revealed that 901 differentially modified m6A genes between MI and control group,of which 537 genes were upregulated,and 364 genes were downregulated.The principal component analysis affirmed that two groups could be distinguished significantly in terms of m6A gene modification.The characteristic sequence of m6A modification was GGACU and mainly concentrated in the coding sequence.According to the conjoint analysis with RNA-seq and MeRIP-seq,119 genes expressed simultaneous m6A modification difference and mRNA expression difference.The Venn diagram exhibited the positive and negative correlation between m6A modification and mRNA expression.Besides,the GO enrichment analysis indicated that the genes with m6A differential modification in MI group were mainly involved in heart development and other processes.qPCR verified that Gbp6 was up-regulated,while Dnaja1 and Dnajb1 were down-regulated.MeRIP-qPCR revealed that the m6A modification level of Hspa1b was downregulated.ConclusionMyocardial infarction induces differential modification of m6A in the mice model.In addition,the genes with m6A modification may be affected by methylation related enzymes,thus participating the pathogenesis of MI by regulating apoptosis and inflammation.