Transcriptome-wide map of N6-methyladenosine (m6A) profiling in coronary artery disease (CAD) with clopidogrel resistance.

BACKGROUND: Clopidogrel resistance profoundly increases the risk of major cardiovascular events in coronary artery disease (CAD) patients. Here, we comprehensively analyse global m6A modification alterations in clopidogrel-resistant (CR) and non-CR patients. METHODS: After RNA isolation, the RNA transcriptome expression (lncRNA, circRNA, and mRNA) was analysed via RNA-seq, and m6A peaks were identified by MeRIP-seq. The altered m6A methylation sites on mRNAs, lncRNAs, and circRNAs were identified, and then, GO and KEGG pathway analyses were performed. Through joint analysis with RNA-seq and MeRIP-seq data, differentially expressed mRNAs harbouring differentially methylated sites were identified. The changes in m6A regulator levels and the abundance of differentially methylated sites were measured by RT-PCR. The identification of m6A-modified RNAs was verified by m6A-IP-qPCR. RESULTS: The expression of 2919 hypermethylated and 2519 hypomethylated mRNAs, 192 hypermethylated and 391 hypomethylated lncRNAs, and 375 hypermethylated and 546 hypomethylated circRNAs was shown to be altered in CR patients. The m6A peaks related to CR indicated lower mark density at the CDS region. Functional enrichment analysis revealed that inflammatory pathways and insulin signalling pathways might be involved in the pathological processes underlying CR. The expression of mRNAs (ST5, KDM6B, GLB1L2, and LSM14B), lncRNAs (MSTRG.13776.1 and ENST00000627981.1), and circRNAs (hsa_circ_0070675_CBC1, hsa-circRNA13011-5_CBC1, and hsa-circRNA6406-3_CBC1) was upregulated in CR patients, while the expression of mRNAs (RPS16 and CREG1), lncRNAs (MSTRG.9215.1), and circRNAs (hsa_circ_0082972_CBC1) was downregulated in CR patients. Moreover, m6A regulators (FTO, YTHDF3, and WTAP) were also differentially expressed. An additional combined analysis of gene expression and m6A peaks revealed that the expression of mRNAs (such as ST5, LYPD2, and RPS16 mRNAs) was significantly altered in the CR patients. CONCLUSION: The expression of m6A regulators, the RNA transcriptome, and the m6A landscape was altered in CR patients. These findings reveal epitranscriptomic regulation in CR patients, which might be novel therapeutic targets in future.

A male-specific association between AGTR1 hypermethylation and coronary heart disease.

The AGTR1 gene encodes angiotensin II receptor type 1, which is involved in cardiovascular diseases such as coronary heart disease (CHD). In the current study, we analyzed AGTR1 methylation level in a Han Chinese population by SYBR green-based quantitative methylation-specific PCR (qMSP). We collected blood samples from 761 CHD patients and 398 non-CHD controls at the Ningbo First Hospital. A data mining analysis was also performed to explore the association between AGTR1 methylation and AGTR1 gene expression, using datasets from the cBioPortal for Cancer Genomics and the Gene Expression Omnibus (GEO) database. Our results showed a significantly higher percentage of methylated reference (PMR) of AGTR1 in male CHD patients compared with male non-CHD controls (median PMR: 2.12% vs. 0.59%, p = 0.037). The data mining analysis showed that AGTR1 expression was significantly increased in human hepatoma HepG2 cells treated with the demethylation agent 5-aza-2'-deoxycytidine (fold = 3.12, p = 0.009). Further data mining analysis using the cholangiocarcinoma (TCGA, PanCancer Atlas) data indicated an inverse association between AGTR1 methylation and AGTR1 expression (r = -0.595, p = 1.29E-04). Overall, our results suggest that AGTR1 methylation is involved in the regulation of AGTR1 gene expression and that AGTR1 hypermethylation is associated with CHD in males. These findings may provide new clues about the pathogenesis of CHD.