miR-449a靶向调控外周血Toll样受体5影响风湿性心脏病发生发展的分子机制研究  

Molecular mechanism of miR-449a targeted regulation of peripheral blood Toll-like receptor 5 on development of rheumatic heart disease

作　　者：李宝亮[1] 苏华[1] 李梦嘉[1] 耿丽娜[1] 马俊帅[1] LI Baoliang;SU Hua;LI Mengjia;GENG Lina;MA Junshuai(Department of Laboratory,the First Affiliated Hospital of Hebei North University,Zhangjiakou 075000,China)

机构地区：[1]河北北方学院附属第一医院检验科,张家口075000

出　　处：《中国免疫学杂志》2022年第17期2057-2063,共7页Chinese Journal of Immunology

基　　金：2019年度河北省医学科学研究课题计划项目(20190866)资助。

摘　　要：目的:研究微小RNA-449a(miR-449a)是否通过靶向外周血中的Toll样受体5(TLR5)影响风湿性心脏病的发生发展。方法:实时定量PCR(qRT-PCR)和Western blot检测风湿性心脏病患者外周血中TLR5和miR-449a的表达水平。在风湿性心脏病心肌细胞中转染miR-449a模拟物或TLR5小干扰RNA si-TLR5。采用qRT-PCR检测miR-449a表达,Western blot分析细胞核相关抗原Ki67(Ki67)、活化的天冬氨酸特异性半胱氨酸蛋白酶-3(Cleaved-caspase-3)、TLR5、磷酸化p65(p-p65)和磷酸化IκBα(p-IκBα)蛋白表达,MTT测定细胞增殖,流式细胞仪评估细胞凋亡,ELISA测定炎症因子IL-1β、TNF-α水平。Starbase靶位点预测软件与双荧光素酶报告基因分析miR-449a与TLR5之间的靶向关系。在风湿性心脏病心肌细胞中共转染miR-449a模拟物和TLR5过表达质粒pcDNA-TLR5,观察细胞增殖、凋亡和NF-κB信号通路活性变化。结果:风湿性心脏病患者外周血中miR-449a的表达水平明显降低,TLR5 mRNA和TLR5蛋白表达水平显著升高(P<0.05)。过表达miR-449a明显提高风湿性心脏病心肌细胞的Ki67蛋白表达水平、细胞存活率,显著降低Cleaved-caspase-3、p-p65、p-IκBα蛋白表达水平、细胞凋亡率和IL-1β、TNF-α水平(P<0.05)。低表达TLR5明显提高风湿性心脏病心肌细胞的Ki67蛋白表达水平、细胞存活率,显著降低Cleaved-caspase-3蛋白表达水平、凋亡率(P<0.05)。miR-449a通过靶向TLR5调控TLR5蛋白的表达。高表达TLR5后,miR-449a对风湿性心脏病心肌细胞增殖、Ki67蛋白表达的促进作用,以及miR-449a对细胞凋亡、Cleaved-caspase-3、p-p65、pIκBα蛋白表达和IL-1β、TNF-α水平的抑制作用被逆转。结论:miR-449a通过靶向TLR5抑制NF-κB信号通路活性,促进风湿性心脏病心肌细胞的增殖与抑制其凋亡。Objective:To investigate whether microRNA-449a(miR-449a) affects the occurrence and development of rheumatic heart disease by targeting Toll-like receptor 5(TLR5)in peripheral blood.Methods:Real-time quantitative PCR(qRTPCR)and Western blot were used to detect the expression levels of TLR5 and miR-449a in peripheral blood of patients with rheumatic heart disease. Transfection of miR-449a mimic or TLR5 small interfering RNA si-TLR5 in rheumatic heart disease myocardial cells.qRT-PCR detected the expression of miR-449a. Western blot was employed to analysis Ki67,Cleaved-caspase-3,TLR5,p-p65 and p-IκBα protein expression,MTT measured cell proliferation,flow cytometry to evaluate apoptosis and ELISA to determine the levels of inflammatory factors IL-1β and TNF-α. Starbase target site prediction software and dual luciferase reporter gene analysis the targeting relationship between miR-449a and TLR5. Co-transfected miR-449a mimics and TLR5 overexpression plasmid pcDNA-TLR5 in rheumatic heart disease myocardial cells to observe changes in cell proliferation,apoptosis and NF-κB signaling pathway activity.Results:The expression level of miR-449a in peripheral blood of patients with rheumatic heart disease was significantly reduced,and the expression levels of TLR5 mRNA and TLR5 protein were significantly increased(P<0.05). Overexpression of miR-449a greatly increased the expression level of Ki67 protein and cell survival rate in rheumatic heart disease cardiomyocytes,and significantly reduced the expression levels of Cleaved-caspase-3,p-p65,p-IκBα protein,the rate of apoptosis and IL-1β,TNF-α levels(P<0.05).Low expression of TLR5 obviously increased the Ki67 protein expression level and cell survival rate of rheumatic heart disease cardiomyocytes,and remarkably reduced the Cleaved-caspase-3 protein expression level and apoptosis rate(P<0.05). miR-449a targeted TLR5 and regulated the expression of TLR5 protein. After high expression of TLR5,miR-449a promoted rheumatic heart disease cardiomyocyte proliferation and

关 键 词：风湿性心脏病 miR-449a TLR5 增殖 凋亡 NF-ΚB信号通路 

分 类 号：R541.2[医药卫生—心血管疾病]