作者：贾敏，李城城，王丽妹，张玲，陈家华，李俊红

单位：

广东省广州市胸科医院综合内科

Units：

General Medicine Ward, Guangzhou Chest Hospital, Guangzhou 510095, China

关键词：

动脉粥样硬化; miRNA-21; SPRY1; 炎症; ERK/NF-κB信号通路;

Keywords：

Atherosclerosis; miRNA-21; SPRY1; Inflammation; ERK/NF-κB signaling pathway

CLC：

DOI：

10.12114/j.issn.1008-5971.2023.00.081

Funds：

广东省医学科学技术研究基金项目（A2021443）；

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Western blot法检测各组SPRY1、磷酸化细胞外调节蛋白激酶（ERK）1/2、磷酸化NF-κB蛋白。(7)将HUVECs分为模拟物对照组、模拟物组、模拟物+U0126组，分别转染模拟物阴性对照（模拟物对照组）、miRNA-21模拟物（模拟物组和模拟物+U0126组），在此基础上模拟物+U0126组使用ERK1/2抑制剂U0126进行处理，转染48 h后均构建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。采用Western blot法检测各组磷酸化ERK1/2、磷酸化NF-κB蛋白。结果 （1）动物实验结果：AS组小鼠miR-21及IL-1β、TNF-α、IL-6 mRNA相对表达量高于对照组（P<0.05）。（2）细胞实验结果：(1)模拟物组miRNA-21及IL-1β、TNF-α、IL-6 mRNA相对表达量高于模拟物对照组（P<0.05）；抑制剂组miRNA-21及IL-1β、TNF-α、IL-6 mRNA相对表达量低于抑制剂对照组（P<0.05）。(2)生物信息学分析结果显示，SPRY1是miRNA-21的潜在靶基因。(3)双荧光素酶报告基因实验结果显示，SPRY1-WT 3’-UTR模拟物组荧光素酶活性低于SPRY1-WT 3’-UTR模拟物对照组（P<0.05）;SPRY1-MUT 3’-UTR模拟物组与SPRY1-MUT 3’-UTR模拟物对照组荧光素酶活性比较，差异无统计学意义（P>0.05）。(4)模拟物组SPRY1 mRNA相对表达量低于模拟物对照组（P<0.05）。(5)模拟物+pcDNA-SPRY1组IL-1β、TNF-α、IL-6 mRNA相对表达量低于模拟物+Vector组（P<0.05）。(6)模拟物组SPRY1蛋白低于模拟物对照组，磷酸化ERK1/2、磷酸化NF-κB蛋白高于模拟物对照组（P<0.05）；模拟物+pcDNA-SPRY1组SPRY1蛋白高于模拟物+Vector组，磷酸化ERK1/2、磷酸化NF-κB蛋白低于模拟物+Vector组（P<0.05）。(7)模拟物+U0126组IL-1β、TNF-α、IL-6 mRNA相对表达量及磷酸化ERK1/2、磷酸化NF-κB蛋白低于模拟物组（P<0.05）。结论 动物实验表明，AS小鼠存在miRNA-21过表达和炎症反应；细胞实验表明，miRNA-21过表达可降低SPRY1，抑制ERK/NF-κB信号通路，进而促进AS内皮细胞炎症反应，故miRNA-21可能是治疗AS的新型分子靶点。

Abstract：

　Objective To investigate the effect of microRNA (miRNA) -21 on the inflammation response ofendothelial cells in atherosclerosis (AS) and its molecular biological mechanism. Methods This experiment was conducted fromJuly 2021 to October 2022. ( 1 ) Animal experiment. Three ApoE-/- mice were included in the AS group and fed with high-fatdiet to construct the AS model. Three C57BL/6 mice were included in the control group and fed with standard diet. The relativeexpression 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. ① HUVECs were divided into mimic group, NC mimic group, inhibitor group and NC inhibitor group, andtransfected with miRNA-21 mimic, mimic negative control, miRNA-21 inhibitor and inhibitor negative control, respectively. AScell model was constructed at 48 h after transfection. The relative expression levels of miRNA-21 and IL-1β, TNF-α, IL-6mRNA in each group were detected by RT-qPCR. ② The bioinformatics analysis was performed online through the of miRNAtarget gene database TargetScan. ③ SPRY1 wild-type plasmid (SPRY1-WT 3'-UTR) and SPRY1 mutant plasmid (SPRY1-MUT3'-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 andmiRNA-21 mimic, SPRY1-WT 3'-UTR and mimic negative control, SPRY1-MUT 3'-UTR and miRNA-21 mimic, SPRY1-MUT3'-UTR and mimic negative control, respectively. Luciferase activity was detected by double luciferase reporter gene assay at 48h after transfection. ④ HUVECs were divided into mimic group and NC mimic group, and transfected with miRNA-21 mimic andmimic negative control, respectively. AS cell model was constructed at 48 h after transfection. The relative expression of SPRY1mRNA in each group was detected by RT-qPCR. ⑤ HUVECs were divided into mimic+Vector group and mimic+pcDNA-SPRY1group, and transfected with miRNA-21 mimic and Vector, miRNA-21 mimic and pcDNA-SPRY1, respectively. The AS cell modelwas constructed at 48 h after transfection. The relative expression levels of IL-1β, TNF-α and IL-6 mRNA in each group weredetected by RT-qPCR. ⑥ HUVECs were divided into mimic group, NC mimic group, mimic+Vector group and mimic+pcDNASPRY1 group, and transfected with miRNA-21 mimic, mimic negative control, miRNA-21 mimic and Vector, miRNA-21mimic and pcDNA-SPRY1, respectively. The AS cell model was constructed at 48 h after transfection. SPRY1, phosphorylatedextracellular regulated protein kinase (ERK) 1/2 and phosphorylated NF-κB were detected by Western blot. ⑦ HUVECs weredivided into NC mimic group, mimic group and mimic+U0126 group, and transfected with mimic negative control (mimic controlgroup) , miRNA-21 mimic (mimic group and mimic+U0126 group) , respectively. On this basis, the mimic+U0126 group wastreated with ERK1/2 inhibitor U0126. At 48 h after transfection, the AS cell model was constructed. The relative expression levelsof IL-1β, TNF-α and IL-6 mRNA in each group were detected by RT-qPCR. Western blot was used to detect phosphorylatedERK1/2 and phosphorylated NF-κB protein in each group. Results （1）Animal experiment results: the relative expressionlevels of miRNA-21, IL-1β, TNF-α, and IL-6 mRNA in AS group were higher than those in control group (P < 0.05) . (2) Cellexperiment results: ① the relative expressions levels of miRNA-21 and IL-1β, TNF-α, IL-6 mRNA in the mimic group werehigher than those in the NC mimic group (P < 0.05) . The relative expression levels of miRNA-21 and IL-1β, TNF-α, IL-6mRNA in the inhibitor group were lower than those in the NC inhibitor group (P < 0.05) . ② Bioinformatics analysis showed thatSPRY1 was a potential target gene of miRNA-21. ③ The results of dual luciferase reporter gene assay showed that the luciferaseactivity in the SPRY1-WT 3'-UTR mimic group was lower than that in the SPRY1-WT 3'-UTR NC mimic group (P < 0.05) .There was no significant difference in luciferase activity between the SPRY1-MUT 3'-UTR mimic group and the SPRY1-MUT 3'-UTR NC mimic group (P > 0.05) . ④ The relative expression level of SPRY1 mRNA in the mimic group was lower than that in theNC mimic group (P < 0.05) . ⑤ The relative mRNA expressions of IL-1β, TNF-α, and IL-6 in the mimic+pcDNA-SPRY1 groupwere lower than those in the mimic+Vector group (P < 0.05) . ⑥ The SPRY1 protein in the mimic group was lower than that in theNC mimic group, and the phosphorylated ERK1/2 and phosphorylated NF-κB protein in the mimic group were higher than those inthe NC mimic group (P < 0.05) . The SPRY1 protein in the mimic+pcDNA-SPRY1 group was higher than that in the mimic+Vectorgroup, and the phosphorylated ERK1/2 and phosphorylated NF-κB protein in the mimic+pcDNA-SPRY1 group were lower thanthose in the mimic+Vector group (P < 0.05) . ⑦ The relative expressions levels of IL-1β, TNF-αand IL-6 mRNA, phosphorylatedERK1/2 and phosphorylated NF-κB protein in the mimic+U0126 group were lower than those in the mimic group (P < 0.05) .Conclusion Animal experiments showed that there was miRNA-21 overexpression and inflammatory response in AS mice. Cellexperiments showed that overexpression of miRNA-21 could reduce SPRY1, inhibit ERK/NF-κB signaling pathway, and promotethe inflammatory response of AS endothelial cells. Therefore, miRNA-21 may be a new molecular target for the treatment of AS.

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