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

Abstract

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.

Keywords: Clopidogrel resistance; Coronary artery disease; RNA transcriptome expression; m6A modification.

Fig. 1

Overview of m6A modification peaks on mRNAs, lncRNAs, and circRNAs in CR and non-CR patients. A–C Venn diagram depicting the common and distinct m6A peaks in mRNAs, lncRNAs, and circRNAs between CR and non-CR patients. D–F The numbers of m6A peaks located on mRNA, lncRNA, and circRNA between CR and non-CR patients

Fig. 2

Overview of m6A peaks located on mRNAs, lncRNAs, and circRNAs in CR and non-CR patients. A Motifs enriched of all the identified m6A peaks in CR and non-CR patients. B Diversity of the m6A peak density in the indicated regions between CR and non-CR patients. C Pie chart revealing the regional distributions of m6A peaks in the RNA transcriptome of CR patients. D Pie chart revealing the regional distributions of m6A peaks in the RNA transcriptome of non-CR patients

Fig. 3

Abnormally m6A-modified mRNAs, lncRNAs, and circRNAs were identified in CR patient samples compared to those in non-CR patients samples. A mRNAs, B lncRNAs, and C circRNAs

Fig. 4

Hierarchical clustering analysis showing the differences in the RNA transcriptome in CR and non-CR patients based on |log₂FC|> 1 and P value < 0.05 criteria. A circRNAs, B lncRNAs, and C mRNAs. In heatmaps, red indicates hypermethylation, and green indicates hypomethylation

Fig. 5

Differences in the chromosomal distribution of differentially methylated m6A sites between CR and non-CR patients. A–C Chromosomal distribution of differentially methylated m6A sites in three different kinds of RNAs. D–F Statistical analysis showed fold change differences in hypermethylated and hypomethylated m6A sites in transcript regions of mRNA, lncRNA, and circRNA in CR versus non-CR patients

Fig. 6

Gene Ontology enrichment analysis of differentially abundant m6A marks between CR and non-CR patients. A circRNAs in CR patients, B lncRNAs in CR patients, C mRNAs in CR patients, D circRNAs in non-CR patients, E lncRNAs in non-CR patients, and F mRNAs in non-CR patients

Fig. 7

KEGG pathway analysis results showing differentially abundant m6A marks between CR and non-CR patients. A circRNAs in CR patients, B circRNAs in non-CR patients, C lncRNAs in CR patients, D lncRNAs in non-CR patients, E mRNAs in CR patients, and F mRNAs in non-CR patients

Fig. 8

Comprehensive analysis of mRNA m6A peaks and their expression between CR and non-CR patients. Scatter plot showing the distribution of mRNAs with both significantly changed m6A and mRNA levels in CR and non-CR patients

Fig. 9

The mRNA expression of m6A regulators in CR and non-CR patients. A–E m6A writers (METTL3, METTL14, METTL4, WTAP, and KIAA1429), F, G m6A erasers (FTO and ALKBH5), and H–L m6A readers (YTHDC1-2 as well as YTHDF1-3)

Fig. 10

Validation of the m6A-enriched genes and candidate loci. A Validation of the m6A-enriched genes as indicated by m6A-immunoprecipitation (IP)-qPCR. B Validation of the expression levels of candidate mRNAs, lncRNAs, and circRNAs as determined by RT-qPCR