The Function And Mechanism Of Circulating MiR-135b-5p And MiR-499a-3p In Atherosclerosis

Atherosclerosis is a chronic and complex disease, involving a variety of physiological, biochemical and pathological changes. Proliferation and migration of endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) are critical processes involved in atherosclerosis. Recent studies have revealed that microRNAs (miRNAs) can be detected in circulating blood with a stable form and the expression profiles differ in many cellular processes associated with coronary artery disease (CAD). However, little is known about their role, especially serum-derived miRNAs, in ECs and VSMCs phenotype modulation during atherosclerosis. We compared the miRNA expressions in serum samples from 13 atherosclerotic CAD patients and 5 healthy control subjects and identified 36 differentially expressed miRNAs. The expression of selected miRNAs (miR-135b-5p and miR-499a-3p) was further validated in 54 serum samples. Interestingly, miR-135b-5p and miR-499a-3p directly regulated a common target gene:myocyte enhancer factor 2C (MEF2C) which plays an important role in modulating cell phenotype of cardiovascular systems. Furthermore, our results indicated that the 2 elevated miRNAs could jointly promote ECs and VSMCs proliferation and migration by repressing MEF2C expression. Together, our findings demonstrated a serum-based miRNA expression profile for atherosclerotic CAD patients, potentially revealing a previously undocumented mechanism for cell proliferation and migration mediated by miR-135b-5p and miR-499a-3p, and might provide novel insights into the role of circulating miRNAs in atherosclerosis pathogenesis.objectiveThis topic mainly studies and the expression of miR-135b-5p and miR-499a-3p in atherosclerosis patients serum, and explore the two microRNAs how to regulate downstream target genes MEF2C.At the same time explore the role of miR-135b-5p and miR-499a-3p in endothelial cells and vascular smooth muscle cells,such as proliferation and migration.Methods1.The acquisition of miRNA in serum:5 ml blood sample were collected from atherosclerosis patients.Using Trizol separation methods, obtained microRNAs in serum.2.Determination the levels of microRNAs in atherosclerosis patient serum:Using the primer design software,we designed miR-135-b-5 p and miR-499-a-3 p specific primers.Through the Real-Time PCR,detected the two microRNAs expression.3.Luciferase reporter assays:Using PCR,we amplificated MEF2C 3’-UTR,and cloning it into the pmirGLO dual luciferase vector.Luciferase activity was measured using the Dual-Luciferase Reporter Assay system.4. Western blot:Respectively transfected the mimics or inhibitors of miR-135b-5p and miR-499a-3p into HUVEC and VSMC.Using protein lysis, cracked cell and collected protein,through the ECL kit detecting protein expression.5.Proliferation experiments:Using the EDU kits,detected the impact of miR-135b-5p and miR-499a-3p on HUVEC and VSMC cell proliferation.6.Migration experiments:Using the Transwell kits and scratch test,detected the impact of miR-135b-5p and miR-499a-3p on HUVEC and VSMC cell migration.7.Immunohistochemical experiments:Using the DAB kits,detected the espression of MEF2C in atherosclerotic plaque and vessel wall.Resultsl.MiRNA profiles in atherosclerosis patients serum:35 abnormal microRNAs were found in atherosclerosis patients serum,including 25 upregelated miRNAs ans10 downregelated miRNAs.2. MiR-135b-5p and miR-499a-3p inhibit the expression of target genes MEF2C: Compared with control group,when increased miR-135b-5p and miR-499a-3p levels,the expression of MEF2C were fell 33.41% and 20.48% respectively.Luciferase experiment shown that fluorescence intensity were fell 45.67% and 26.39% respectively, while there are no change in MEF2C mRNA.3.MiR-135b-5p and miR-499a-3p promote proliferation and migration: compared with control,when upregulate the miR-135b-5p and miR-499a-3p level, HUVEC and VSMC proliferation rate and migration rate are significantly increased.4.MEF2C is lower expression in atherosclerotic plaque:Compared with the vascular wall, MEF2C was lower expression in atherosclerotic plaques.ConclusionsThe present study demonstrated a miRNA expression profile in atherosclerosis patients serum and both miR-135b-5p and miR-499a-3p regulate the expression of MEF2C together,and promote HUVEC and VSMC proliferation and migration though MEF2C.