Abstract
<jats:p>DNA methylation is a key epigenetic mechanism regulating the expression of genes involved in numerous developmental and pathological processes. However, the contribution of DNA methylation of microRNA genes to atherosclerosis remains poorly understood. In this study, we profiled DNA methylation patterns of both the regulatory elements and gene bodies of five microRNA genes (MIR10B, MIR21, MIR100, MIR127, and MIR143) in vascular tissues and paired peripheral blood cells (PBC) of 92 patients with advanced carotid atherosclerosis and 32 PBC of control participants by targeted bisulfite sequencing. We identified distinct tissue-specific DNA methylation patterns for all five microRNA genes in patients with advanced carotid atherosclerosis. The regulatory regions of MIR10B, MIR127, and MIR100 were moderately hypomethylated in carotid atherosclerotic plaques compared with intact vascular tissues. We further integrated our findings with lab-internal and publicly available epigenome-wide methylation datasets and evaluated the influence of vascular and blood cell composition using computational deconvolution approaches. After adjustment for cellular heterogeneity in vascular tissues, DNA methylation at CpG sites in MIR100 and MIR127 remained independently associated with atherosclerosis. Increased DNA methylation at a single CpG site (chr11:122025143, GRCh37/hg19) located within the MIR100 E-box region was associated with metabolic syndrome. Moreover, DNA methylation levels of MIR10B, MIR21, and MIR127 in atherosclerotic plaques were linked with indicators of histological instability and history of acute cerebrovascular events. In peripheral blood, we observed moderate hypomethylation of the regulatory regions of MIR10B, MIR21, and the MIR100 E-box region in patients compared with the control group. However, only the MIR10B remained robust against blood cell composition. In blood, MIR10B and MIR143 methylation correlated with lipid metabolism and carotid stenosis, while the MIR21 CpG island showed strong blood-plaque concordance, confirming its potential as a surrogate biomarker. Overall, advanced carotid atherosclerosis is characterized by specific tissue-altering DNA methylation patterns of microRNA genes, where alterations mainly occur in the regulatory regions, predominantly featuring hypomethylation. The results underscore the complex, cell- and tissue-specific nature of DNA methylation of microRNA genes in both the regulatory elements and gene bodies in vascular tissue and blood, highlighting the critical need to decipher these intricate epigenetic landscapes to identify reliable, robust biomarkers for assessing plaque instability and cardiovascular risk.</jats:p>