摘要
目的研究微小RNA(microRNA,miRNA)miR-25-3p对心肌成纤维细胞纤维化表型的调控及机制。方法血管紧张素Ⅱ(AngⅡ)灌注小鼠构建心肌纤维化模型。用miRNA表达谱芯片检测纤维化的小鼠心肌中表达水平有差异的miRNA。原代分离法获得C57BL/6小鼠心肌成纤维细胞(mCF),建立AngⅡ处理mCF的心肌纤维化细胞模型。mCF转染miR-25-3p mimic后检测纤维化相关基因的表达。双荧光素酶报告基因实验验证miR-25-3p与B细胞易位基因2(BTG2)的3′端非翻译区(3′UTR)的结合作用。放线菌素D实验验证BTG2对超氧化物歧化酶2(SOD2)稳定性的作用。结果miR-25-3p在AngⅡ灌注诱导的小鼠心肌和AngⅡ处理的mCF中表达升高。转染miR-25-3p mimic可使mCF中的Ⅰ型胶原α1(COL1A1)、Ⅲ型胶原α1(COL3A1)和α-平滑肌肌动蛋白(α-SMA)基因表达显著增加。双荧光素酶报告基因实验和功能实验证实BTG2是miR-25-3p的下游靶基因,并参与介导miR-25-3p促进mCF中纤维化相关基因表达的作用。机制研究证实BTG2可通过增加SOD2 m RNA的稳定性而上调其表达。结论miR-25-3p通过抑制BTG2的表达来下调SOD2的水平进而增强纤维化相关基因的表达,发挥促进心肌纤维化的作用。
Aim To investigate the effect of microRNA miR-25-3p on fibrosis-related gene expression in cardiac fibroblasts and mechanism involved.Methods A mouse model of cardiac fibrosis induced by angiotensinⅡ(AngⅡ)i nfusion was constructed,and the differentially expressed miRNAs in the fibrotic mouse myocardium were detected by miRNA chip array.Primary isolation and culture of C57BL/6 mouse cardiac fibroblasts(mCF)were performed,and a cell model of myocardial fibrosis was established based on AngⅡ-treated mCF.The effect of miR-25-3p mimic on the expression of fibrosis-related genes in mCF was studied.The dual luciferase reporter gene assay was performed to verify the binding of miR-25-3p on the 3′-untranslated region(3′UTR)of the B-cell translocation gene 2(BTG2)gene.Actinomycin D experiment was performed to detect the effect of BTG2 on the stability of superoxide dismutase 2(SOD2)mRNA in mCF.Results Expression of miR-25-3p was increased in the myocardium of AngⅡ-infused mice and AngⅡ-treated mCF.miR-25-3p could enhance the expression of fibrosis-related genes,including collagen typeⅠalpha 1 chain(COL1A1),collagen typeⅢalpha 1 chain(COL3A1)andα-smooth muscle actin(α-SMA)in mCF.BTG2 was confirmed to be a target gene of miR-25-3p,and BTG2 mediated the effect of miR-25-3p on promoting fibrosis-related gene expression in mCF.In addition,BTG2 could enhance the expression of SOD2 by increasing the stability of SOD2 mRNA in mCF.Conclusion MiR-25-3p inhibited the level of SOD2 through targeting BTG2 in mCF,resulting in enhancing fibrosis-related gene expression and promoting myocardial fibrosis.
作者
赵安职
郭晶
陈丽文
黄智琪
陈泽润
朱杰宁
张梦珍
张铭
徐金东
单志新
ZHAO Anzhi;GUO Jing;CHEN Liwen;HUANG Zhiqi;CHEN Zerun;ZHU Jiening;ZHANG Mengzhen;ZHANG Ming;XU Jindong;SHAN Zhixin(School of Pharmacy,Southern Medical University,Guangzhou,Guangdong 510515,China;School of Medicine,South China University of Technology,Guangzhou,Guangdong 510006,China;Guangdong Cardiovascular Institute,Guangzhou,Guangdong 510080,China;School of Biology and Biological Engineering,South China University of Technology,Guangzhou,Guangdong 510006,China;The Second School of Clinical Medicine,Southern Medical University,Guangzhou,Guangdong 510280,China;Guangdong Provincial Key Laboratory of Clinical Pharmacology&Guangdong Provincial People’s Hospital&Guangdong Academy of Medical Sciences,Guangzhou,Guangdong 510080,China)
出处
《中国动脉硬化杂志》
CAS
2022年第4期328-334,351,共8页
Chinese Journal of Arteriosclerosis
基金
国家自然科学基金项目(81770264、82070254)
广东省自然科学基金项目(2021A1515011554)
广东省医学科研基金项目(B2020215)
广州市科技计划项目(202002030013、202002030039、202102080093)。
