MiR-126 on mice with coronary artery disease by targeting S1PR2.

J-L Fan, L Zhang, X-H Bo

European review for medical and pharmacological sciences 2020 Jan

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To screen the differentially expressed micro ribonucleic acids (miRNAs) in the serum of coronary atherosclerosis patients, and to investigate their possible mechanisms of action. The differentially expressed serum miRNAs were screened from 3 coronary artery disease (CAD) patients and 3 healthy controls using miRNA expression profiles, which were verified using low-throughput quantitative Reverse Transcription-Polymerase Chain Reaction (RT-qPCR) assay. 60 apolipoprotein E (ApoE)-/- mice were divided into model group, agomir-126 group, agomir-control (con) group, and antagomir-126 group using a random number table. They were fed with high-fat diets (21% fat and 0.15% cholesterol) ad libitum for 15 weeks to establish the mouse model of CAD. Then, hematoxylin and eosin (HE) staining was applied to detect the impact of miR-126 expression level on the tissue morphology in the thoracic aortic region. The influences of miR-126 expression level on the secretion levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and IL-10 were determined via enzyme-linked immunosorbent assay (ELISA). Western blotting assay was performed to examine the effects of miR-126 expression level on the expression levels of nuclear factor-kappa B (NF-κB) and vascular cell adhesion molecule-1 (VACM-1) in the tissues of the thoracic aortic region of the mice. The correlation between miR-126 expression level and sphingosine-1-phosphate receptor 2 (S1PR2) in the serum of CAD patients and animal models was analyzed by the Pearson correlation coefficient method. The targets of miR-126 were predicted using the bioinformatics method, and the direct targets were verified through investigations. Western blotting assay and ELISA were adopted to detect the impacts of miR-126 expression level on the expression and secretion levels of TNF-α, IL-1β, and IL-10 in S1P + oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVECs). Lentivirus-small hairpin RNA (shRNA) was utilized to knock down the expression level of S1RP2 to determine whether miR-126 affected the increase in the inflammation level in S1P + ox-LDL-induced HUVECs by targeting S1RP2. Compared with those in control group, 4 miRNAs (miR-126, miR-206, miR-4297, and miR-3646) in the serum of CAD patients exhibited the most significant expression differences, which increased by 6.72, 7.11, 13.57, and 21.22 times, respectively. The verification results of low-throughput RT-qPCR assay indicated that there were remarkable changes in the expression levels of the 4 selected miRNAs with differential expressions in comparison with those in control group, displaying statistically significant differences (p<0.01). The results of HE staining manifested that the coronary atherosclerotic plaques were reduced markedly in agomir-126 group, while notably more coronary atherosclerotic plaques were formed in the thoracic aortic region in antagomir-126 group. Meanwhile, the elevated expression level of miR-126 evidently lowered the expressions of serum TNF-α and IL-1β, but significantly increased the expression of IL-10 in the mouse model of CAD. According to the analysis results of the Pearson correlation coefficient method, the miR-126 expression level was negatively correlated with S1PR2 expression level in the serum of both CAD patients and animal models (r=-0.6123, r=-5.37). It was shown in bioinformatics prediction and luciferase reporter gene assay that miR-126 negatively regulated the S1PR2 expression by targeting the 3' untranslated region (UTR) of S1PR2 messenger RNA (mRNA). In the in vitro inflammation model, the increased expression level of miR-126 could relieve the inflammation in cells induced by S1P + ox-LDL. Based on the results of both Western blotting assay and ELISA, the differences in the expression and secretion levels of TNF-α, IL-1β, and IL-10, as well as the expression levels of signaling molecules of the NF-κB signaling pathway, in the cells were not statistically significant among miR-126 mimic treatment group, sh-S1PR2 group, and miR-126 mimic + sh-S1PR2 group, indicating that miR-126 affects the inflammation level in HUVECs by targeting S1PR2. MiR-126 represses the progression of coronary atherosclerosis in the mice by binding to S1PR2. The results of this research may propose a new mechanism of miR-126 in exerting its therapeutic effects and possess potential value for the treatment of CAD in the future.

Citation

J-L Fan, L Zhang, X-H Bo. MiR-126 on mice with coronary artery disease by targeting S1PR2. European review for medical and pharmacological sciences. 2020 Jan;24(2):893-904