MicroRNA-99b-5p通过抑制成纤维细胞生长因子21表达促进心肌细胞肥大  

MicroRNA-99b-5p Aggravates Cardiomyocyte Hypertrophy by Down-regulating Fgf21

作　　者：陈丽文[1] 郭晶 陈泽润 朱杰宁[1] 徐金东[1] 郭惠明[1] 单志新[1,2,3] 王晟 CHEN Li-wen;GUO Jing;CHEN Ze-run;ZHU Jie-ning;XU Jin-dong;GUO Hui-ming;SHAN Zhi-xin;WANG Sheng(Guangdong Cardiovascular Institute//Guangdong Provincial People’s Hospital//Guangdong Academy of Medical Sciences,Guangzhou 510080,China;School of Medicine,South China University of Technology,Guangzhou 510006,China;The Second School of Clinical Medicine,Southern Medical University,Guangzhou 510280,China.)

机构地区：[1]广东省心血管病研究所//广东省人民医院//广东省医学科学院,广东广州510080 [2]华南理工大学医学院,广东广州510006 [3]南方医科大学第二临床医学院,广东广州510280

出　　处：《中山大学学报（医学科学版）》2022年第2期192-202,共11页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：国家自然科学基金(82070254,81770264);广州市科技计划项目(202002030039,202102080093);广东省自然科学基金(2021A1515011554);广东省人民医院心血管专项(2020XXG003)。

摘　　要：【目的】研究微小RNA microRNA-99b-5p(miR-99b-5p)对心肌细胞肥大表型的调节作用及机制。【方法】实时荧光定量PCR(RT-qPCR)检测人心肌组织(包括健康对照者、心力衰竭患者心肌组织)以及横向主动脉缩窄(TAC)手术小鼠心肌中miR-99b-5p的表达水平。血管紧张素Ⅱ(AngⅡ)处理原代乳小鼠心肌细胞(NMVCs)后,利用鬼笔环肽染色呈现心肌细胞大小,RT-qPCR检测miR-99b-5p表达水平。利用RT-qPCR及蛋白质免疫印迹法检测转染miR-99b-5p mimic对NMVCs中肥大相关基因及成纤维细胞生长因子21(Fgf21)表达的影响。双萤光素酶报告实验验证miR-99b-5p与Fgf21基因3′端非翻译区(3’UTR)的结合作用。利用腺病毒介导在NMVCs中过表达Fgf21及超氧化物歧化酶2(Sod2),并利用小干扰RNA(siRNA)敲低NMVCs中Fgf21和Sod2表达,研究Fgf21/Sod2轴是否介导miR-99b-5p对心肌细胞肥大表型的调节作用。【结果】MiR-99b-5p在心衰患者心肌组织、TAC术后的小鼠心肌组织及在AngⅡ处理的心肌细胞中均表达上调(P<0.01)。MiR-99b-5p可促进NMVCs中与肥大相关基因的表达(P<0.01)。双萤光素酶报告基因实验证实miR-99b-5p与Fgf21基因的3’UTR存在结合作用,并且miR 99b-5p可抑制Fgf21及其下游基因Sod2表达(P<0.05)。在NMVCs中过表达Fgf21或Sod2均可有效逆转miR 99b-5p的促心肌细胞肥大作用(P<0.05)。【结论】MiR-99b-5p在肥厚心肌中表达增加,并通过Fgf21/Sod2轴发挥促进心肌细胞肥大的作用。【Objective】To explore the role of microRNA-99b-5p(miR-99b-5p)in cardiomyocyte hypertrophy and the related mechanism involved.【Methods】The expression of miR-99b-5p in myocardium was detected by real-time quan⁃titative PCR(RT-qPCR)in the myocardium of patients with heart failure(HF)and healthy controls,as well as in the myo⁃cardium of mouse model of transverse aortic restriction(TAC)-induced cardiac hypertrophy.Phalloidin-iFluor 647 stain⁃ing was used to show the size of neonatal mouse ventricular cardiomyocytes(NMVCs)after AngⅡtreatment.MiR-99b-5p expression was determined by RT-qPCR in AngⅡ-treated NMVCs.After transfection with miR-99b-5p mimic,the ex⁃pression of cardiac hypertrophy-associated genes and Fgf21 in NMVCs was detected by RT-qPCR and Western blot as⁃say,respectively.We identified the interaction between miR-99b-5p and the 3’UTR of Fgf21 mRNA by dual luciferase reporter assay.The recombinant Ffg21 adenovirus(rAd-Fgf21)and rAd-Sod2 were used to infect NMVCs,and the expres⁃sion ofβ-MHC,ANP,FGF21 and SOD2 was detected by Western blot assay.We knocked down Fgf21 and Sod2 in NMVCs to investigate the role of FGF21/Sod2 axis in miR-99b-5p-regulated NMVC hypertrophy.【Results】MiR-99b-5p expression was elevated in the myocardium of HF patients and TAC-operated mice,and in AngⅡ-treated NMVCs(P<0.01,respectively).MiR-99b-5p promoted the expression of hypertrophy-related genes in NMVCs(P<0.01).Results of dual luciferase reporter gene assay revealed the interaction between miR-99b-5p and Fgf21 mRNA.MiR-99b-5p down regulated the expression of Fgf21 and the down-stream gene of Sod2(P<0.01).Overexpression of FGF21 or SOD2 could inhibit NMVC hypertrophy and effectively reversed the pro-hypertrophy effect of miR-99b-5p on NMVCs(P<0.05,re⁃spectively).【Conclusion】MiR-99b-5p is up-regulated in the hypertrophic myocardium and enhances cardiomyocyte hy⁃pertrophy via suppressing Fgf21/Sod2 axis.

关 键 词：心肌细胞肥大 微小RNA-99-5p 成纤维细胞生长因子21 

分 类 号：R363.2[医药卫生—病理学]