Peripheral Blood Mononuclear Cell-Derived miR-664a-3p is Associated with Plaque Burden and Necrotic Core Characteristics in Coronary Artery Disease Across Two Independent Populations

Background: Stability of the atherosclerotic plaque in coronary artery disease (CAD) is determined by features such as total plaque burden and necrotic core volume. Since invasive procedures are required to evaluate plaque stability, we tested whether the peripheral blood mononuclear cell (PBMC) microRNA (miR) signature could correlate with measures of plaque stability and thus serve as a non-invasive biomarker. Method: Patients from two distinct geographical locations in India were recruited to the study (Site 1: CAD=19, non-CAD=5; Site 2: CAD=12, non-CAD=7) and underwent invasive intravascular ultrasound with virtual histology to assess plaque burden and necrotic core volume. RNA from PBMCs of these patients was subjected to unbiased sequencing. Differential miR expression evaluated by DESeq2 and assessed for co-relationship with plaque stability. miR target gene prediction was performed using multiple databases, and Enrichr was used for enrichment analysis. Results: Unbiased RNA sequencing identified miR-664a-3p to be significantly downregulated in CAD patients from both sites (Site 1: log2FC=-1.02, p=0.0033 & Site 2: log2FC=-1.04, p=0.0007). miR-664a-3p expression was inversely correlated with plaque burden and necrotic core volume. Receiver operating characteristic (ROC) analysis of miR-664a-3p showed significant discriminative performance in the CAD cohort, with AUC values of 0.842 (Site 1) and 0.881 (Site 2). miR664a-3p target prediction and pathway enrichment analysis revealed selective enrichment of inflammatory signaling pathways, such as IL-17 and TNF, suggesting an association between PBMC pro-inflammatory response and plaque vulnerability. Conclusion: miR-664a-3p is downregulated in CAD patients and inversely correlates with measures of plaque stability, with potential as a biomarker for identifying patients at risk of CAD progression and plaque instability. Keywords: Coronary artery disease, peripheral blood mononuclear cells (PBMCs), microRNA, atherosclerotic plaque, necrotic core, plaque burden, biomarkers.