Liu Shidong, Liu Hongxu, Bai Qiyuan, Wei Zhili, Zhao Pengying, Chen Hao, Song Bing, Yu Cuntao
The First Clinical Medical College of Lanzhou University, Lanzhou, China.
Department of Cardiovascular Surgery, First Hospital of Lanzhou University, Lanzhou, China.
Mol Biol Rep. 2025 Aug 26;52(1):850. doi: 10.1007/s11033-025-10940-2.
This study aimed to investigate the role and potential mechanisms of N6-methyladenosine (m6A) methylation in a mouse model of transverse aortic constriction (TAC)-induced cardiac fibrosis using MeRIP-seq. A TAC-induced cardiac fibrosis mouse model was established, and cardiac function and structural parameters were assessed by echocardiography four weeks post-surgery. The global m6A methylation levels in myocardial tissues were evaluated using Dot blot analysis, and the expression levels of m6A-modifying enzymes (METTL3, METTL14, ALKBH5, FTO) were detected by qPCR and Western blot. Additionally, methylated RNA immunoprecipitation sequencing (MeRIP-Seq) was performed to identify differentially methylated sites, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses to determine the biological functions and signaling pathways of m6A-methylated genes. The results showed that TAC surgery successfully induced a cardiac fibrosis model in mice, as indicated by significantly reduced EF and LVEF and increased LVIDd and LVIDs. Moreover, m6A methylation levels were elevated in myocardial tissues of TAC mice, accompanied by upregulation of METTL3 and METTL14 expressions and downregulation of ALKBH5 and FTO expression. MeRIP-seq revealed that m6A peaks were primarily enriched in the 3' UTR regions, with 1,466 differentially methylated sites identified between TAC and sham groups, including 717 hypermethylated and 749 hypomethylated sites. Functional enrichment analyses showed that these differentially methylated genes were involved in various biological processes, including signal transduction, transcriptional regulation, and ion channel activity, and were associated with pathways such as type 2 diabetes mellitus, signaling pathways regulating pluripotency of stem cells, and insulin signaling. Thus, our findings suggested that m6A methylation played a significant role in TAC-induced cardiac fibrosis by regulating key genes involved in myocardial remodeling and functional impairment.
本研究旨在利用甲基化RNA免疫沉淀测序（MeRIP-seq），在横断主动脉缩窄（TAC）诱导的心脏纤维化小鼠模型中，研究N6-甲基腺苷（m6A）甲基化的作用及潜在机制。建立了TAC诱导的心脏纤维化小鼠模型，并在术后四周通过超声心动图评估心脏功能和结构参数。使用斑点印迹分析评估心肌组织中的整体m6A甲基化水平，并通过qPCR和蛋白质免疫印迹检测m6A修饰酶（METTL3、METTL14、ALKBH5、FTO）的表达水平。此外，进行甲基化RNA免疫沉淀测序（MeRIP-Seq）以鉴定差异甲基化位点，随后进行基因本体（GO）和京都基因与基因组百科全书（KEGG）通路富集分析，以确定m6A甲基化基因的生物学功能和信号通路。结果显示，TAC手术成功诱导了小鼠心脏纤维化模型，表现为射血分数（EF）和左心室射血分数（LVEF）显著降低，左心室内径舒张末期（LVIDd）和收缩末期（LVIDs）增加。此外，TAC小鼠心肌组织中的m6A甲基化水平升高，同时伴有METTL3和METTL14表达上调以及ALKBH5和FTO表达下调。MeRIP-seq显示，m6A峰主要富集在3'非翻译区（UTR）区域，在TAC组和假手术组之间鉴定出1466个差异甲基化位点，其中包括717个高甲基化位点和749个低甲基化位点。功能富集分析表明，这些差异甲基化基因参与了各种生物学过程，包括信号转导、转录调控和离子通道活性，并与2型糖尿病、调节干细胞多能性的信号通路和胰岛素信号通路等途径相关。因此，我们的研究结果表明，m6A甲基化通过调节参与心肌重塑和功能损伤的关键基因，在TAC诱导的心脏纤维化中发挥重要作用。
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