| Objective Currently,the most common method is to use stents to treat coronary atherosclerotic heart disease,but implantation of stents can lead to damage to the vessel wall,endothelial dysfunction and high differentiation of vascular smooth muscle cells.Abnormal proliferation or migration of Vascular Smooth Muscle Cells is an important factor leading to the development of atherosclerosis(AS)and in-stent restenosis(IRS).This study was to explore the role and molecular mechanism of miR-378a-5p in regulating VSMCs function.Methods(1)Apolipoprotein E-deficient mice(Apolipoprotein E,ApoE-/-)were given a high-fat diet to construct an atherosclerosis model group,healthy basal-fed C57BL/6J mice were used to construct a control group,obtaining heart blood from both groups and using Reverse Transcription-Polymerase Chain Reaction(RT-PCR)to examine the expression of miR-378a-5p in two groups.(2)Obtain Clinical blood samples from patients with clinical stent restenosis and healthy individuals,and detect the expression of miR-378a-5p in the blood.(3)Transfection of miR-378a-5p mimic and inhibitor into VSMCs using transient transfection techniques by transfection reagent lipo2000 and then detection of expression of miR-378a-5p.(4)The proliferation of VSMCs was evaluated by EdU assay and Cell Counting Kit-8(CCK-8)assay,respectively.(5)Scratch test and transwell assay were applied to evaluate VSMCs migration.(6)Western blot was used to detect the expression of p21 and CDK1 proteins.(7)TargetScan software was used to predict target genes,and bio-informatics analysis was used to explore the biological functions of candidate genes.(8)RNA binding protein immunoprecipitation(RIP)and immunofluorescence technique(IF)were used to analyze the interaction of miRNA with protein and binding properties,localization and expression of miR-378a-5p to downstream target CDK1.Results(1)RT-PCR results show that the expression level of miR-378a-5p was up-regulated in both atherosclerosis mice and human stent restenosis blood samples,the difference was statistically significant(P<0.05).(2)The expression of miR-378a-5p wasincreased in VSMC stimulated by Platelet Derived Growth Factor(PDGF)compared with NC group,the difference was statistically significant(P<0.05).(3)CCK-8 and EdU assays showed that cells were more proliferative than NC after 24 h of transfection of miR-378a-5p mimics,and the difference was statistically significant(P<0.05);For the effect of cell migration,we observed that miR-378a-5p significantly promoted cell migration compared with NC,the difference was statistically significant(P<0.05).(4)Western blot shows that inhibition of CDK1 expression promotes proliferation and migration of VSMC,the expression of phenotype-transformed protein smMHC2 on VSMC transfected with siCDK1 was decreased.(5)CDK1 is the functional target of miR-378a-5p in VSMC,In immunofluorescence experiments,the expression of CDK1 is decreased in miR-378a-5p mimic transfected VSMC,while the miR-378a-5p inhibitor group has opposite results,and the fluorescence intensity is enhanced.The difference is statistically significant.Significance(P<0.05);RIP assay showed that the efficiency of enrichment of CDK1 in plasmids expressing miR-378a-5p was significantly higher than that in the non-miR-378a-5p group,the difference was statistically significant(P<0.05).(6)miR-378a-5p acts on CDK1 and p21 to regulate the proliferation and migration of VSMC.Conclusions MiR-378a-5p is involved in the regulation of proliferation and migration of VSMC;miR-378a-5p regulates VSMC proliferation and migration by targeting CDK1 / p21 signaling pathway;miR-378a-5p has the potential ability to prevent and treat atherosclerosis and stent restenosis. |
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