Baseline characteristics of controls and CA patients
We recruited a total of 52 CA patients, including 36 males, with an average age of (56.79 ± 6.81) years. In addition, 25 healthy volunteers were recruited as normal controls, including 15 males, with an average age of (54.23 ± 9.18) years. Table 2 lists the baseline characteristics of CA patients and controls. The comparisons indicated that there was no statistical difference in gender and age between the two groups (both P > 0.05), while the laboratory parameters (triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (HDL-C) and high-density lipoprotein cholesterol (HDL-C) were significantly different (all P < 0.05).
miR-126 and miR-223 are lowly expressed in the serum of CA patients
First, we analyzed the differential expression profiles of miRNA in the serum of 6 CA patients and 6 controls by miRNA microarray sequencing. With the setting screening conditions, we found that there were 430 differentially expressed miRNAs, of which 197 were up-regulated and 233 were down-regulated (Fig. 1A). The heatmap in Fig. 1B shows the first differentially expressed 30 miRNAs. Subsequently, we further performed RT-qPCR to detect the top 10 differentially expressed miRNAs (miR-384, miR-219a, miR-126, miR-101, miR-577, miR-135a, miR-223, miR-148, miR-137 and miR-149) in 52 CA patients and 25 healthy volunteers. The obtained results were consistent with the trend of microarray analysis (Fig. 1C). Afterwards, we analyzed the predictive efficacy of these 10 differentially expressed miRNAs in CA patients by ROC curve. We found that miR-126 and miR-223, which had lower expression in the serum of CA patients, had the largest AUC (Fig. 1D).
miR-223 and miR-126 have a good predictive effect on plaque instability in CA patients
Next, these 52 CA patients were allocated into plaque stable (PS, n = 23) and plaque unstable (PU, n = 29) based on contrast enhanced ultrasound test results, ultrasound test results, and clinical test history (symptomatic or asymptomatic). The comparison revealed that the low expression levels of miR-223 and miR-126 were detected in the serum of the PU group versus the PS group (both P < 0.05, Fig. 2A). Meanwhile, the ROC curve was adopted to analyze the predictive power of the expression levels of miR-223 and miR-126 on the plaque stability of CA patients, and findings suggested that the expression levels of miR-223 and miR-126 had high predictive power (AUC: greater than 0.75) on instability plaque of CA patients (Fig. 2B).
miR-126 and miR-223 are negatively correlated with plaque instability factors in CA patients
Subsequently, we detected the serum levels of IL-6, MMP1, MMP9 and MCP1 in CA patients by ELISA, and results suggested that serum levels of IL-6, MMP1, MMP9 and MCP1 in PU patients were higher than those in PS patients (Fig. 3A). Further, the correlations between the expression levels of miR-223 and miR-126 in serum of CA patients and these factors were analyzed by Pearson's correlation analysis, we found that the serum levels of miR-126 and miR-223 were negatively correlated with IL-6, MMP1, MMP9 and MCP1 (Fig. 3B-E). In addition, we also found that the expression levels of miR-126 and miR-223 were negatively correlated with the plaque thickness in CA patients (both P < 0.05) (Fig. 3F). The results can preliminarily indicate that miR-126 and miR-223 have a certain effect on maintaining the stability of atherosclerotic plaque.
miR-126 and miR-223 target COX2
With the aim to explore the possible downstream molecular mechanisms of miR-126 and miR-223 in maintaining the stability of atherosclerotic plaques, we predicted the downstream targeting mRNAs of miR-126 and miR-223 through StarBase and TargetScan databases, respectively, and there existed an intersection: COX2 (Fig. 4A). Next, luciferase activity experiments indicated that co-transfection of miR-126 mimic or miR-223 mimic with COX2-WT could reduce the luciferase activity in HEK293T cells (Fig. 4B, C), confirming that COX2 was miR-126’ and miR-223’ direct target gene. Based on the aforementioned, we further examined the expression level of COX2 in the serum of CA patients and normal volunteers. Results revealed that high expression level of COX2 was found in the serum of CA patients relative to normal controls (Fig. 4D), and its level in the PU patients was also higher than that in the PS patients (both P < 0.05) (Fig. 4E). Further Pearson’s correlation analysis suggested that the expression levels of miR-126 and miR-223 in serum of CA patients were negatively correlated with the expression level of COX2 (Fig. 4F).
COX2 expression level is positively correlated with plaque instability factors in CA patients
Furthermore, we analyzed the correlation between the expression level of COX2 and the plaque stability-related factors in the serum of CA patients through Pearson's correlation analysis. We found that the expression level of COX2 was positively correlated with levels of IL-6, MMP1, MMP9, MCP1 as well as plaque thickness (Fig. 5A-E). This result indicates that COX2 can promote the expression of IL-6, thereby promoting the secretion of MCP1 by macrophages, thus inducing carotid artery epithelial cells to analyze MMPs, thereby promoting the plaque instability of CA.