Circulating MiRNA As Biomarkers For Coronary Plaque Instability And Its Mechanisms

Background:Coronary heart disease is one of the most common cause of death in the world, and coronary plaque instability is a major cause of acute coronary events. So there is of great significance of early identification and intervention of instable plaque in reducing the incidence and mortality of cardiovascular events. But until now there is no ideal instable plaque identifying markers, so the development of noninvasive and easy screening method is important on the explorion of the unstable plaque rupture mechanism. MicroRNAs (miRNAs, miRs) is a class of endogenous evolutionary highly conserved non-coding single stranded RNA. Circulating miRNA refers to free miRNA in plasma or serum, with a stable and highly conserved sequence, tissue or pathology specific expression, and is gradually used as noninvasive diagnostic and prognostic biomarkers. But research on circulating miRNA as biomarkers for coronary plaque instability is still unknown, and the role of miRNA on plaque rupture is not very clear. So, looking for specific miRNA in the peripHeral blood is helpful on early and accurate prediction for plaque rupture, and providing new models and targets for the treatment of atherosclerosis and prevention.Objectives:In the present study, we would like to evaluate the:(1)miRNA whose contents were different in the peripHeral blood of patients with non-calcified plaque, and the probability of circulating miRNAs as a biomarker in identifying unstable plaque using the contents changes.(2)The Correlation of target genes and instable plaques associated protein markers, and the molecular mechanism of miRNA in the role of plaque rupture. Methods:Outpatients of Fuwai hospital, from September2011to February2013were divided into three groups by64-slice spiral CT examination and the clinical symptoms of ACS.5mL fasting serum samples were collected, and were stored at-80℃.12cases were selected in initial screening.11kinds of miRNAs (miR-1, miR-16, miR-21, miR-133a, miR-133b, miR-126, miR-143, miR-145, miR-155, miR-221, miR-222) associated with plaque instability were detected using TaqMan qRT-PCR method by relative quantitative analysis, if the miRNA’s expression in non-plaque group more than1.5fold than the normal group, a further detection in expanded sample size (n=185cases;61in non-calcified,62in calcified and62in control) was continued. The level of MMP-9and MMP-2in serum were determined by ELISA. Overexpressing the target miRNA in human macrophages, and detecting its impact on protein and mRNA levels of target gene use of western and RT-PCR method and the influence on expression and secretion of MMP-2and MMP-9. The luciferase reporter assay was used to investigate the miRNA target for validation. The siRNA technique was used for the knockdown of target gene in macrophages to further investigate the effect of transfection of miRNA on MMP-2and MMP-9.The results were analyzed using SPSS17.0. P<0.05was considered statistically significant.Results:The screening results showed6kinds of miRNA (miR-1, miR-16, miR-21, miR-126, miR-155, miR-221, miR-222) expressed in non-plaque group more than1.5fold than the normal group; in expanded size samples showed there were differences in the levels of miR-16, miR-21, miR-126, miR-155and miR-221among three groups. There were differences in the levels of miR-16and miR-21between non-calcified plaque group and calcified group as well as the control. The areas under the ROC curve for the combination of miR-16, miR-21, MMP-2and MMP-9miR-16to predict non-calcified plaques was0.868. Serum miR-16level was positively correlated with miR-21, and serum miR-16level was positively correlated with serum MMP-9. In atherosclerosis related laboratory biochemical indicators HDL showed positive correlation with miR-16. The odd ratio (OR) of miR-16and miR-21for predicting non-calcified plaques was2.397and5.837respectively. We also found that in macrophages the level of miR-16and miR-21in non-calcified plaque patients’macrophages was significantly higher than that in calcified groups and control. miR-16and miR-21could promote the expression and secretion of MMP-9in human macrophages. RECK is a novel target of miR-16, and was involved in the MMP-9up-regulation induced by miR-16and miR-21in human macrophages. miR-16and miR-21significantly increased the protein level of MMP-9via suppressing the target gene of RECK to degrade the collagen in the cap.Conclusions:Serum miR-16and miR-21may be a predictor of coronary unstable plaque and they contributes to the plaque instability under the mechanism that promoting the expression and secretion of MMP-9in macrophages by suppressing target gene RECK.