MiR-125a-5p靶向胰岛素样生长因子1受体调节川崎病中内皮细胞的增殖和迁移  

MiR-125a-5p targets insulin-like growth factor 1 receptor to regulate endothelial cell proliferation and migration in Kawasaki disease

作　　者：吕爱婷 何坤 李莹莹[1] 候维纳 孙琪青[1] LÜAiting;HE Kun;LI Yingying;HOU Weina;SUN Qiqing(Department of Cardiology,Children’s Hospital Affiliated to Zhengzhou University/Henan Children’s Hospital/Zhengzhou Children’s Hospital,Zhengzhou 450000,China)

机构地区：[1]郑州大学附属儿童医院/河南省儿童医院/郑州儿童医院心内科,郑州450000

出　　处：《临床与病理杂志》2023年第2期211-220,共10页Journal of Clinical and Pathological Research

基　　金：河南省医学科技攻关计划联合共建项目(LHGJ20200647);郑州儿童医院联合共建项目(2018020695)。

摘　　要：目的:探讨miR-125a-5p靶向胰岛素样生长因子1受体(insulin-like growth factor 1 receptor,IGF1R)对川崎病内皮细胞增殖与迁移的影响。方法:培养人冠状动脉内皮细胞(human coronary artery endothelial cells,HCAECs),将细胞分为对照组(20%正常人血清)、20%川崎病血清组(20%川崎病患儿血清)、20%川崎病血清+抵制剂(inhibitor)正常对照(normal control,NC)组、20%川崎病血清+miR-125a-5p inhibitor组、20%川崎病血清+miR-125a-5p inhibitor+siRNA组、20%川崎病血清+miR-125a-5p inhibitor+si IGF1R组,除对照组添加20%正常人血清外,其余各组均添加20%川崎病患儿血清培养。定量RT-PCR法测定miR-125a-5p、IGF1R mRNA表达水平,蛋白质印迹法测定IGF1R蛋白表达;细胞计数试剂盒8(cell counting kit 8,CCK-8)法测定HCAECs细胞增殖能力;Transwell法测定细胞迁移能力;蛋白质印迹法测定Ki-67、血管内皮钙黏蛋白(vascular endothelial-cadherin,VE-cad)、p120连环蛋白(p120-catenin,p120ctn)水平;构建野生型IGF1R重组质粒(pGL4-IGF1R-WT)和突变型IGF1R重组质粒(pGL4-IGF1R-MUT),将HCAECs细胞分为mimic-NC+pGL4-IGF1R-WT组、miR-125a-5p mimic+pGL4-IGF1R-WT组、mimic-NC+pGL4-IGF1R-MUT组、miR-125a-5p mimic+pGL4-IGF1R-MUT组,双荧光素酶报告实验测定miR-125a-5p与IGF1R mRNA的水平。结果:与对照组相比,20%川崎病血清组HCAECs细胞5-乙炔基-2’-脱氧尿嘧啶核苷(5-ethynyl-2’-deoxyuridine,EdU)结合率、miR-125a-5p水平、VE-cad、p120ctn表达均升高(均P<0.05),IGF1R mRNA与蛋白水平、HCAECs细胞光密度(optical density,OD)值、Ki-67均降低(均P<0.05);与20%川崎病血清组相比,20%川崎病血清+miR-125a-5p inhibitor组HCAECs细胞EdU结合率、miR-125a-5p水平、VE-cad、p120ctn表达均降低,IGF1R mRNA水平与蛋白水平、HCAECs细胞OD值、Ki-67均升高(均P<0.05);与20%川崎病血清+miR-125a-5p inhibitor组相比,20%川崎病血清+miR-125a-5p inhibitor+si IGF1R组HCAECs细胞EdU结合率miR-125a-5p水平、VE-cad、p120ctn表�Objective:To investigate the effect of miR-125a-5p targeting on insulin-like growth factor 1 receptor(IGF1R)on proliferation and migration of endothelial cells in Kawasaki disease.Methods:Human coronary artery endothelial cells(HCAECs)were cultured,and the cells were divided into a control group(20%normal human serum),a 20%Kawasaki disease serum group(20%Kawasaki disease children serum),a 20%Kawasaki disease serum+inhibitor normal control(NC)group,a 20%Kawasaki disease serum+miR-125a-5p inhibitor group,a 20%Kawasaki disease serum+miR-125a-5p inhibitor+siRNA group,and a 20%Kawasaki disease serum+miR-125a-5p inhibitor+si IGF1R group,except for the control group supplemented with 20%normal human serum.The other groups were supplemented with 20%serum from children with Kawasaki disease.Quantitative RT-PCR method was used to determine the expression levels of miR-125a-5p and IGF1R mRNA.Western blot was used to determine IGF1R protein expression;cell counting kit 8(CCK-8)method was used to determine the proliferation ability of HCAECs;transwell method was used to measure cell migration ability;Western blot was used to determine the levels of Ki-67,vascular endothelial-cadherin(VEcad),and p120-catenin(p120ctn);Wild type IGF1R recombinant plasmid(pGL4-IGF1R-WT)and mutant type IGF1R recombinant plasmid(pGL4-IGF1R-MUT)were constructed.HCAECs cells were divided into mimic-NC+pGL4-IGF1R-WT group,miR-125 a-5p mimic+pGL4-IGF1R-WT group,mimic-NC+pGL4-IGF1R MUT group,miR-125a-5p mimic+pGL4-IGF1R-MUT group,a dual-luciferase reporter assay was used to determine the level between miR-125a-5p and IGF1R mRNA.Results:Compared with the control group,the 5-ethynyl-2’-deoxyuridine(EdU)binding rate,the level of miR-125a-5p,and the expression of VEcad and p120ctn in HCAECs in the 20%Kawasaki disease serum group increased(all P<0.05),the mRNA and protein levels of IGF1R,and the optical density(OD)value of HCAECs,and Ki-67 decreased(all P<0.05);compared with the 20%Kawasaki disease serum group,the 20%Kawasaki disease serum+miR-125a-5p inhi

关 键 词：miR-125a-5p 胰岛素样生长因子1受体 川崎病 内皮细胞 增殖 迁移 

分 类 号：R725.4[医药卫生—儿科]