摘要
目的探讨微小RNA(miRNA)-21对动脉粥样硬化(AS)内皮细胞炎症反应的影响及其分子生物学机制。方法本实验时间为2021年7月至2022年10月。(1)动物实验。将3只ApoE^(-/-)小鼠纳入AS组,给予高脂饲料饲养以构建AS模型;将3只C57BL/6小鼠纳入对照组,给予标准饲料饲养。采用RT-qPCR检测AS组和对照组小鼠miRNA-21及IL-1β、TNF-α、IL-6 mRNA相对表达量。(2)细胞实验。①将人脐静脉内皮细胞(HUVECs)分为模拟物组、模拟物对照组、抑制剂组和抑制剂对照组,分别转染miRNA-21模拟物、模拟物阴性对照、miRNA-21抑制剂、抑制剂阴性对照,转染48 h后构建AS细胞模型。采用RT-qPCR检测各组miRNA-21及IL-1β、TNF-α、IL-6 mRNA相对表达量。②通过miRNA靶基因数据库TargetScan在线进行生物信息学分析。③构建SPRY1野生型质粒(SPRY1-WT 3'-UTR)和SPRY1突变型质粒(SPRY1-MUT 3'-UTR)。将HUVECs分为SPRY1-WT 3'-UTR模拟物组、SPRY1-WT 3'-UTR模拟物对照组、SPRY1-MUT 3'-UTR模拟物组、SPRY1-MUT 3'-UTR模拟物对照组,分别转染SPRY1-WT 3'-UTR和miRNA-21模拟物、SPRY1-WT 3'-UTR和模拟物阴性对照、SPRY1-MUT 3'-UTR和miRNA-21模拟物、SPRY1-MUT 3'-UTR和模拟物阴性对照,转染48 h后采用双荧光素酶报告基因实验检测其荧光素酶活性。④将HUVECs分为模拟物组和模拟物对照组,分别转染miRNA-21模拟物、模拟物阴性对照,转染48 h后构建AS细胞模型。采用RT-qPCR检测各组SPRY1 mRNA相对表达量。⑤将HUVECs分为模拟物+Vector组、模拟物+pcDNA-SPRY1组,分别转染miRNA-21模拟物和Vector、miRNA-21模拟物和pcDNA-SPRY1,转染48 h后构建AS细胞模型。采用RT-qPCR检测各组IL-1β、TNF-α、IL-6 mRNA相对表达量。⑥将HUVECs分为模拟物组、模拟物对照组、模拟物+Vector组、模拟物+pcDNA-SPRY1组,分别转染miRNA-21模拟物、模拟物阴性对照、miRNA-21模拟物和Vector、miRNA-21模拟物和pcDNA-SPRY1,转染48 h后构建AS细胞模型。采用Weste
Objective To investigate the effect of microRNA(miRNA)-21 on the inflammation response of endothelial cells in atherosclerosis(AS)and its molecular biological mechanism.Methods This experiment was conducted from July 2021 to October 2022.(1)Animal experiment.Three ApoE^(-/-)mice were included in the AS group and fed with high-fat diet to construct the AS model.Three C57BL/6 mice were included in the control group and fed with standard diet.The relative expression levels of miRNA-21 and IL-1β,TNF-α,IL-6 mRNA in AS group and control group were detected by RT-qPCR.(2)Cell experiment.①HUVECs were divided into mimic group,NC mimic group,inhibitor group and NC inhibitor group,and transfected with miRNA-21 mimic,mimic negative control,miRNA-21 inhibitor and inhibitor negative control,respectively.AS cell model was constructed at 48 h after transfection.The relative expression levels of miRNA-21 and IL-1β,TNF-α,IL-6 mRNA in each group were detected by RT-qPCR.②The bioinformatics analysis was performed online through the of miRNA target gene database TargetScan.③SPRY1 wild-type plasmid(SPRY1-WT 3'-UTR)and SPRY1 mutant plasmid(SPRY1-MUT 3'-UTR)were constructed.HUVECs were divided into SPRY1-WT 3'-UTR mimic group,SPRY1-WT 3'-UTR NC mimic group,SPRY1-MUT 3'-UTR mimic group and SPRY1-MUT 3'-UTR NC mimic group,and transfected with SPRY1-WT 3'-UTR and miRNA-21 mimic,SPRY1-WT 3'-UTR and mimic negative control,SPRY1-MUT 3'-UTR and miRNA-21 mimic,SPRY1-MUT 3'-UTR and mimic negative control,respectively.Luciferase activity was detected by double luciferase reporter gene assay at 48 h after transfection.④HUVECs were divided into mimic group and NC mimic group,and transfected with miRNA-21 mimic and mimic negative control,respectively.AS cell model was constructed at 48 h after transfection.The relative expression of SPRY1 mRNA in each group was detected by RT-qPCR.⑤HUVECs were divided into mimic+Vector group and mimic+pcDNA-SPRY1 group,and transfected with miRNA-21 mimic and Vector,miRNA-21 mimic and pcDNA-SPRY1,respec
作者
贾敏
李城城
王丽妹
张玲
陈家华
李俊红
JIA Min;LI Chengcheng;WANG Limei;ZHANG Ling;CHEN Jiahua;LI Junhong(General Medicine Ward,Guangzhou Chest Hospital,Guangzhou 510095,China)
出处
《实用心脑肺血管病杂志》
2023年第4期89-95,共7页
Practical Journal of Cardiac Cerebral Pneumal and Vascular Disease
基金
广东省医学科学技术研究基金项目(A2021443)。
