Effect Of MiRNA-26a On The Induction Of Human Coronary Artery Endothelial Cells By Serum From Patients With Kawasaki Disease

Objective: To investigate the effect of miRNA-26a-5p on coronary artery vascular endothelial cells(HCAECs)in Kawasaki disease.Methods: Samples from 20 children with confirmed KD and 20 normal children were collected and divided into experience and control groups.The q RT-PCR method to detect the expression level of miRNA-26a-5p.The Elisa method detects the expression levels of TNF-α,IL-6 inflammatory factors and EZH2 protein expression levels in the two groups.HCAECs were used as the model in vitro,and the serum of children with confirmed KD and normal children were used to stimulate HCAECs in vitro,which were divided into KD group and HC group.Then,according to the serum of children with KD,HCAECs were induced in vitro and transfected with miRNA-26a-5p inhibitor and negative control,they were divided into KDi group and KDi-C group.The q RT-PCR method detects the expression levels of miRNA-26a-5p,EZH2,TNF-α,and IL-6,and the Western blot method detects EZH2,TNF-α,IL-6,Caspase-3,Caspase-9,Bax,Bcl-2 protein expression level;Flow cytometry,transwell migration assay,Scratch migration assay and Matirgel colloid lumen formation were performed to detect apoptosis,metastatic invasion,and cellular vasculature in each group of cells.The dual luciferase reporter system was used to identify the targeting relationship between miRNA-26a-5p and EZH2.Results: In the serum of children with KD,the miRNA-26a-5p,TNF-α and IL-6 expression level was significantly higher than that of the control group(P<0.01);while the EZH2 expression level was significantly lower than that of the control group(P<0.01).After induction of HCAECs cells with KD serum,the expression levels of miRNA-26a-5p,cell apoptosis,lumen formation,apoptosis-related proteins Caspase-3,Caspase-9,Bax,and inflammatory cytokines TNF-α,IL-6 in KD group were significantly higher than those in HC group(P<0.01).However,the expression level of apoptosis-related protein Bcl-2,cell migration and invasion ability were significantly lower than those in HC group(P<0.01).After transfection into miRNA-26a-5p inhibitors,the expression levels of miRNA-26a-5p decreased(P< 0.05)and significantly inhibited apoptosis,lumen formation,and inflammatory factors TNF-αand IL-6(P<0.01).In contrast,the ability of cell migration and cell invasion was significantly increased after transfection into miRNA-26a-5p inhibitor in contrast to miRNA-26a-5p expression(P<0.05).The luciferase activity assay verified that miRNA-26a-5p could target binding to EZH2.Conclusions: miRNA-26a-5p regulate the expression of the EZH2 gene through target binding and affect the metastasis,invasion,apoptosis,and lumen formation of HCAECs,as well as the expression levels of TNF-α and IL-6 inflammatory factors.Therefore,miRNA-26a-5p may be a marker of KD coronary artery injury and may serve as its potential therapeutic target.