背景:动脉粥样硬化(AS)是心血管疾病进展过程中的关键病理事件。它在动脉壁上表现出纤维脂肪病变,缺乏有效的治疗。N6-甲基腺苷(m6A)是真核生物RNA最常见的修饰形式,在调节心血管疾病的发生发展中起重要作用。然而,m6A修饰在AS中的作用尚不清楚。因此,在这项研究中,我们探讨了m6A修饰在AS中的转录组分布及其潜在机制。
方法:甲基化定量试剂盒用于检测AS小鼠主动脉中的整体m6A水平。Western印迹用于分析甲基转移酶的蛋白质水平。甲基化RNA免疫沉淀与下一代测序(MeRIP-seq)和RNA测序(RNA-seq)用于获得AS小鼠主动脉中m6A亚甲基图的首次转录组范围分析,其次是生物信息学分析。qRT-PCR和MeRIP-qRT-PCR用于测量靶基因中的mRNA和m6A水平。
结果:在AS小鼠的主动脉中,甲基转移酶METTL3的整体m6A和蛋白质水平显着增加。然而,脱甲基酶ALKBH5的蛋白水平显著降低。通过MeRIP-seq,我们获得了AS和对照小鼠的m6A甲基化图谱。总的来说,AS组检测到26,918个与13,744个基因相关的m6A峰,而对照组中检测到与13,283个基因相关的26,157个m6A峰。峰主要出现在靠近具有RRACH基序的终止密码子的编码序列(CDS)区域中。此外,功能富集分析表明,含m6A的基因在AS相关途径中显著富集。有趣的是,通过对MeRIP-seq和RNA-seq数据的整合分析,发现m6A甲基化丰度与基因表达水平呈负相关.在m6A修饰的基因中,低甲基化但上调(低上调)基因Fabp5可能是AS的潜在生物标志物。
结论:我们的研究首次提供了全转录组m6A甲基化,以确定m6A修饰与AS进展之间的关联。本研究为进一步探讨AS的发病机制奠定了基础,为AS的治疗提供了新的方向。
BACKGROUND: Atherosclerosis (AS) is a critical pathological event during the progression of cardiovascular diseases. It exhibits fibrofatty lesions on the arterial wall and lacks effective treatment. N6-methyladenosine (m6A) is the most common modification of eukaryotic RNA and plays an important role in regulating the development and progression of cardiovascular diseases. However, the role of m6A modification in AS remains largely unknown. Therefore, in this study, we explored the transcriptome distribution of m6A modification in AS and its potential mechanism.
METHODS: Methylation Quantification Kit was used to detect the global m6A levels in the aorta of AS mice. Western blot was used to analyze the protein level of methyltransferases. Methylated RNA immunoprecipitation with next-generation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) were used to obtain the first transcriptome range analysis of the m6A methylene map in the aorta of AS mice, followed by bioinformatics analysis. qRT-PCR and MeRIP-qRT-PCR were used to measure the mRNA and m6A levels in target genes.
RESULTS: The global m6A and protein levels of methyltransferase METTL3 were significantly increased in the aorta of AS mice. However, the protein level of demethylase ALKBH5 was significantly decreased. Through MeRIP-seq, we obtained m6A methylation maps in AS and control mice. In total, 26,918 m6A peaks associated with 13,744 genes were detected in AS group, whereas 26,157 m6A peaks associated with 13,283 genes were detected in the control group. Peaks mainly appeared in the coding sequence (CDS) regions close to the stop codon with the RRACH motif. Moreover, functional enrichment analysis demonstrated that m6A-containing genes were significantly enriched in AS-relevant pathways. Interestingly, a negative correlation between m6A methylation abundance and gene expression level was found through the integrated analysis of MeRIP-seq and RNA-seq data. Among the m6A-modified genes, a hypo-methylated but up-regulated (hypo-up) gene
Fabp5 may be a potential biomarker of AS.
CONCLUSIONS: Our study provides transcriptome-wide m6A methylation for the first time to determine the association between m6A modification and AS progression. Our study lays a foundation for further exploring the pathogenesis of AS and provides a new direction for the treatment of AS.