微小核糖核酸-31对大肿瘤抑制因子2及心肌细胞肥大的调控作用  

Regulatory Effects of miRNA-31 on LATS2 and Cardiomyocyte Hypertrophy in Rat's Cardiomyocyte in vitro

作　　者：曾俊义[1] 张婉[1] 丁露[1] 魏云峰[1] 郑泽琪[1] 文通[2] 付勇南[2] ZENG Jun-yi ZHANG Wan DING Lu WEI Yun-feng ZHENG Ze-qi WEN Tong FU Yong-nan(Institute of Hypertension at Jiangxi Province, The First Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi, Chin)

机构地区：[1]南昌大学第一附属医院江西省高血压病研究所,江西省南昌市330006 [2]南昌大学第一附属医院心血管内科,江西省南昌市330006

出　　处：《中国循环杂志》2017年第2期177-182,共6页Chinese Circulation Journal

基　　金：江西省卫生计生委科技计划(20161019)

摘　　要：目的:通过下调肥大心肌细胞中微小核糖核酸(miR)-31的表达,观察miR-31对大肿瘤抑制因子2(LATS2)及心肌细胞肥大的调控作用。方法:体外分离大鼠心肌细胞并连续培养10天,按干预条件不同将心肌细胞分为4组:空白对照组、单纯血管紧张素Ⅱ(AngⅡ)干预组、miR-31抑制物慢病毒感染组、阴性病毒感染组。培养第8天实时荧光定量逆转录聚合酶链式反应(qRT-PCR)检测各组心肌细胞miR-31、LATS2及肥大基因心房钠尿肽(ANP)、β-肌球蛋白重链(β-MHC)表达。培养第10天心肌细胞荧光探针染色观察心肌细胞形态变化。蛋白免疫印迹(Western blot)检测LATS2蛋白表达变化。双荧光素酶报告基因质粒转染293T细胞并检测荧光素酶活性,鉴定miR-3l对LATS2的靶向作用。结果:与空白对照组相比,单纯AngⅡ干预组miR-31、心肌肥大基因ANP、β-MHC表达水平均显著上升(P<0.05),心肌细胞相对表面积明显增大(P<0.05),而LATS2基因及蛋白表达明显下调(P<0.05);与单纯AngⅡ干预组比较,miR-31抑制物慢病毒感染组miR-31、心肌肥大基因ANP、β-MHC表达明显下调(P<0.05),心肌细胞相对表面积减小(P<0.05),而LATS2在基因水平略有上升,蛋白水平则显著上调(P<0.05)。双荧光素酶报告基因检测显示TRAF6-3'UTR+miR-146b相对荧光素酶活性较TRAF6-3'UTR+miR-NC显著性下降(P<0.01),LATS2-3'UTR+miR-31相对荧光素酶活性较LATS2-3'UTR-NC+miR-31显著性降低(P<0.01),差异均有统计学意义。结论:下调肥大心肌细胞miR-31表达水平可一定程度逆转心肌细胞肥大,miR-31靶向作用LATS2参与调控心肌细胞的肥大。Objective： To observe the regulatory effects of miRNA-31 （miR-31） on LATS2 and cardiomyocyte hypertrophy via down-regulating miR-31 expression in rat＇s cardiomyocytesin vitro. Methods： Rat＇s cardiomyocytes were isolated and cultured for 10 daysin vitro, according to different intervention methods, the cells were divided into 4 groups：①Blank control group,②AngII intervention group,③Lentivirus with miR-31 inhibitor infection group,④Negative lentivirus infection group. On day-8, gene expressions of MiR-31, LATS2, cardiac hypertrophy ANP and β-MHC were examined by qRT-PCR; on day-10, cell morphology was observed by fluorescence staining. LATS2 protein expression was examined by Western blot analysis. Dual luciferase reporter plasmids were transfected into 293T cells, then luciferase activity was detected to identify the targeting effect of miR-31 on LATS2. Results： Compared with Blank control group, AngII intervention group showed increased gene expressions of miR31, cardiac hypertrophy ANP and β-MHC,P〈0.05, enlarged cardiomyocyte surface,P〈0.05; while decreased gene and proteinexpressions of LATS2,P〈0.05. Compared with AngII intervention group, Lentivirus with miR-31 inhibitor infection group had down-regulated expressions of miR31, cardiac hypertrophy ANP and β-MHC,P〈0.05, reduced cardiomyocyte surface, P〈0.05; while slightly increased LATS2 gene expression and obviously increased protein expression,P〈0.05. Dual luciferase reporter assay presented that relative luciferase activity of TRAF6-3＇ UTR＋miR-146b was significantly decreased than TRAF6-3＇ UTR＋miR-NC,P〈0.01 and relative luciferase activity of LATS2-3＇ UTR＋ miR-31 was signiifcantly reduced than LATS2-3＇ UTR-NC＋miR-31,P〈0.01. Conclusion： Cardiomyocytes hypertrophy could be reversed at certain degree by down-regulating miR-31; the targeting effect of miR-31 on LATS2 was involved in cardiomyocyte hypertrophyregulation.

关 键 词：核糖核酸类 肿瘤抑制蛋白质类 肌细胞 心脏 

分 类 号：R54[医药卫生—心血管疾病]