Differential Expression of microRNAs in Severely Calcified Carotid Plaques

Abstract

We investigated whether microRNA (miRNA) alteration is related to the presence of calcification in carotid plaques. We classified 10 plaques from carotid endarterectomy patients into high- and low-calcified plaques based on Agatston calcium scores. A microarray analysis for miRNA profiles was performed, with validation by a miRNA quantitative real-time polymerase chain reaction (qRT-PCR). The miRNA microarray identified 697 probes; 657 of them were downregulated. We selected the genes that satisfied total gene signal (TGS) >50, |Log2 ratio| > 1 and ≥1 of the following: (1) false discovery rate (FDR) <.05 in the comparison of mean values of logarithmic transformed signals between the groups; (2) .05 ≤ FDR < .1 and showing either high or median for context score+ in miRSearch among the 72 carefully selected genes related to angiogenesis or calcification; and (3) FDR<.1 in the comparison of 10 individual sets of high- and low-calcified plaques. The expression of miRNA validated by qRT-PCR revealed a significant downregulation of hsa-miR-4530, hsa-miR133b, and hsa-miR-1-3p. A Spearman's rank correlation analysis revealed that the logarithmic TGSs for the microarray of hsa-miR-4530 and hsa-miR-133b were significantly inversely correlated with the carotid plaques' calcium scores, and the delta Cq values for the qRT-PCR showed a direct association. In high-calcified carotid plaques, a specific profile for miRNA may be identified, and the expressions of hsa-miR-4530 and hsa-miR-133b had inverse correlations with the calcium score in the plaques, suggesting that miRNAs may play a modulating role in calcified plaques and plaque stability.