摘要
【目的】探讨微小RNA microRNA-199a-3p(miR-199a-3p)对心肌细胞肥大的调控作用及其作用靶基因。【方法】原代分离培养C57BL/6乳小鼠心肌细胞(NMVC),转染miR-199a-3p模拟物(mimic)和视网膜母细胞瘤转录辅阻遏子1(Rb-1)siRNA分别来增加NMVC中miR-199a-3p和抑制Rb-1的水平。双荧光素酶报告基因实验检测miR-199a-3p与潜在靶基因Rb-1 3′端非翻译区(3′-UTR)的结合作用。FITC-鬼笔环肽染色检测乳小鼠心肌细胞表面积。RT-qPCR和Western blot检测miR-199a-3p,Rb-1和心肌细胞肥大相关基因的表达。【结果】①过表达miR-199a-3p可明显增加NMVC中的肥厚相关基因Nppa,Acta1,Myh7表达;②双荧光素酶报告基因实验结果显示miR-199a-3p与Rb-1 3′UTR具有特异结合作用;miR-199a-3p可在转录后水平抑制Rb-1的表达;③过表达miR-199a-3p或抑制Rb-1表达均能一致性地增加心肌细胞表面积和心肌细胞肥大相关基因表达,并促进E2f2进入NMVC细胞核。【结论】miR-199a-3p通过抑制Rb-1表达,促进了E2f2进入细胞核来发挥促进NMVC肥大的作用。
【Objective】To investigate the role and the potential target of miR-199a-3p in mouse cardiac hypertrophy.【Methods】Neonatal mouse ventricular cardiomyocytes(NMVC)were isolated from the hearts of 0-3-day-old newborn C57BL/6 mice.MiR-199a-3p mimic and retinoblastoma transcriptional corepressor 1(Rb-1)siRNA were transfected in.to NMVC to elevate the level of miR-199a-3p and inhibit Rb-1 expression,respectively.NMVC were stained with FITCphalloidin solution to determine the size of NMVC.Dual luciferase reporter assay was performed to identify the interaction between miR-199a-3p and the 3’UTR of Rb-1.mRNA and protein expression of cardiac hypertrophy associated genes were determined by RT-qPCR and Western blotting assay,respectively.【Results】(1)Over-expression of miR-199a-3p could significantly enhance the expression of cardiac hypertrophy-related genes in NMVC;(2)Dual-luciferase reporter assay results verified that miR-199a-3p can interact with the 3’UTR of Rb-1.MiR-199a-3p could suppress Rb-1 ex?pression at the post-transcriptional level;(3)Functionally,miR-199a-3p mimic,consistent with Rb-1 siRNA,could increase cell size and the expression of Nppa,Acta1 and Myh7 in NMVC,and promote the nuclear translocation of E2f2 in NMVC.【Conclusions】MiR-199a-3p promotes the entry of E2f2 into the nucleus through inhibiting the expression of Rb-1,contributing to cardiomyocyte hypertrophy.
作者
杨静
胡志琴
朱杰宁
李晖
符永恒
袁淑菁
潘蓉
张梦珍
单志新
YANG Jing;HU Zhi-qin;ZHU Jie-ning;LI Hui;FU Yong-heng;YUAN Shu-jing;PAN Rong;ZHANG Meng-zhen;SHAN Zhi-xin(School of Medicine,South China University of Technology,Guangzhou 510006,China;Research Center of Medical Sciences//Guangdong Cardiovascular Institute//Guangdong General Hospital//Guangdong Academy of Medical Sciences,Guangzhou 510080,China;School of Pharmacy,Southern Medical University,Guangzhou 510515,China)
出处
《中山大学学报（医学版）》
CAS
CSCD
北大核心
2019年第1期23-30,共8页
Journal of Sun Yat-Sen University:Medical Sciences
基金
国家自然科学基金(81470439
91649109
81770264)
广州市科技计划项目(201804010045)
