circRNA_100395通过结合miR-31-5p抑制心肌细胞肥大相关基因的表达  

circRNA_100395 inhibits cardiac hypertrophy-related gene expression via binding to miR-31-5p

作　　者：郭晶 陈丽文 温艺红 黄智琪 陈泽润 刘彦俊 朱杰宁 方咸宏[2] 徐金东 单志新 GUO Jing;CHEN Li-wen;WEN Yi-hong;HUANG Zhi-qi;CHEN Ze-run;LIU Yan-jun;ZHU Jie-ning;FANG Xian-hong;XU Jin-dong;SHAN Zhi-xin(School of Medicine,South China University of Technology,Guangzhou 510006,China;Guangdong Cardiovascular In-stitute,Guangzhou 510080,China;School of Biology and Biological Engineering,South China University of Technology,Guangzhou 510006,China;The Second School of Clinical Medicine,Southern Medical University,Guangzhou 510280,China;Guangdong Provincial Key Laboratory of Clinical Pharmacology,Guangdong Provincial People's Hospital,Guang-dong Academy of Medical Sciences,Guangzhou 510080,China)

机构地区：[1]华南理工大学医学院,广东广州510006 [2]广东省心血管病研究所,广东广州510080 [3]华南理工大学生物科学与工程学院,广东广州510006 [4]南方医科大学第二临床医学院,广东广州510280 [5]广东省临床药理学重点实验室,广东省人民医院,广东省医学科学院,广东广州510080

出　　处：《中国病理生理杂志》2022年第1期56-64,共9页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.82070254,No.81770264);广州市科技计划项目(No.202002030013,No.202002030039,No.202102080093);广东省自然科学基金资助项目(No.2021A1515011554)。

摘　　要：目的:探讨环状RNA(circRNA)_100395对心肌细胞肥大表型的影响及其作用机制。方法:通过RT-qPCR检测健康器官捐献者(n=8)与心衰患者(n=14)心肌组织中circRNA_100395及其宿主基因Kelch蛋白样家族成员20(KLHL20)的表达水平。原代分离、培养乳小鼠心室肌细胞(NMVCs),分别感染对照病毒(rAd-GFP)和circRNA_100395重组腺病毒(rAd-circRNA_100395)24 h,探究circRNA_100395对心肌肥大相关的β-肌球蛋白重链(β-MHC)、骨骼肌α-肌动蛋白(ACTA1)及心房钠尿肽(ANP)表达的影响。采用鬼笔环肽染色检测circRNA_100395对血管紧张素II(AngII)处理的NMVCs肥大反应的影响。通过双萤光素酶报告基因实验及RNApull-down实验验证微小RNA-31-5p(miR-31-5p)与circRNA_100395的结合作用。通过转染miR-31-5p mimic至NMVCs中,检测miR-31-5p对circRNA_100395抑制心肌肥大相关基因表达的影响。结果:在心衰患者心肌组织中,circRNA_100395表达水平及其宿主基因KLHL20的mRNA表达水平显著增加(P<0.05)。过表达circRNA_100395可以降低NMVCs中心肌肥大相关基因Myh7(编码β-MHC蛋白)、Acta1及Anp的表达,抑制AngII的促NMVCs肥大反应(P<0.01)。双萤光素酶报告基因实验和RNApull-down实验结果表明miR-31-5p与circRNA_100395存在相互结合作用。过表达miR-31-5p可减弱circRNA_100395对心肌细胞肥大的抑制作用。结论:circRNA_100395通过结合miR-31-5p发挥抑制心肌细胞肥大的作用。AIM:To investigate the effect of circular RNA(circRNA)_100395 on hypertrophy phenotype of cardiomyocytes and the mechanism involved.METHODS:Expression of circRNA_100395 and its host gene Kelch pro⁃tein-like family member 20(KLHL20)was detected by RT-qPCR in the myocardium of healthy organ donors(n=8)and heart failure(HF)patients(n=14).Neonatal mouse ventricular cardiomyocytes(NMVCs)were infected with GFP adeno⁃virus(rAd-GFP)and recombinant circRNA_100395 adenovirus(rAd-circRNA_100395)for 24 h to explore the effect of circRNA_100395 on the expression ofβ-myosin heavy chain(β-MHC),skeletal muscleα-actin(ACTA1)and atrial natriuretic peptide(ANP).The effect of circRNA_100395 on the size of NMVCs exposed to angiotensin II(Ang II)treat⁃ment was evaluated by Phalloidin-iFluor 647 staining.The binding of microRNA-31-5p(miR-31-5p)with circRNA_100395 was verified by dual-luciferase reporter assay and RNA pull-down assay.RT-qPCR and Western blot were used to detect the effect of miR-31-5p on hypertrophy-related gene expression in NMVCs with over-expression of circRNA_100395.RESULTS:The expression of circRNA_100395 and the mRNA expression of its host gene KLHL20 were signifi⁃cantly increased in the myocardium of HF patients(P<0.05).Over-expression of circRNA_100395 significantly inhibited the expression myocardial hypertrophy-related genes,including Myh7(encodingβ-MHC protein),Acta1 and Anp,in NMVCs,and suppressed Ang II-induced increase in the size of NMVCs(P<0.01).The results of dual-luciferase reporter assay and RNA pull-down assay confirmed the interaction between miR-31-5p and circRNA_100395.Over-expression of miR-31-5p attenuated the inhibitory effect of circRNA_100395 on cardiomyocyte hypertrophy.CONCLUSION:circRNA_100395 inhibits hypertrophy-related gene expression through binding to miR-31-5p in NMVCs.

关 键 词：心肌细胞肥大 环状RNA_100395 微小RNA-31-5p 

分 类 号：R542.2[医药卫生—心血管疾病] R363.2[医药卫生—内科学]