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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 effect of miR-31 on LATS2 and cardiomyocyte hypertrophy by down-regulating the expression of miRNA-31 in hypertrophic cardiomyocytes. Methods: Cardiomyocytes were isolated and cultured continuously for 10 days in vitro. Cardiomyocytes were divided into 4 groups according to different intervention conditions: blank control group, Ang Ⅱ intervention group, miR-31 inhibitor lentivirus infection group , Negative virus infection group. The expression of miR-31, LATS2 and atrial natriuretic peptide (ANP) and beta-myosin heavy chain (beta-myosin heavy chain) in cardiomyocytes of each group were detected by real-time quantitative reverse transcription polymerase chain reaction MHC) expression. On day 10, cardiomyocytes were stained with fluorescent probe to observe the morphological changes of cardiomyocytes. Western blot analysis of LATS2 protein expression changes. The luciferase reporter gene plasmid was transfected into 293T cells and luciferase activity was detected to identify the targeting effect of miR-3l on LATS2. Results: Compared with the blank control group, the expression levels of ANP and β-MHC significantly increased (P <0.05) and the relative surface area of myocardial cells increased significantly (P <0.05) The expression of LATS2 gene and protein was significantly down-regulated (P <0.05). Compared with AngⅡ alone group, the expression of miR-31 and cardiac hypertrophy genes ANP and β-MHC in miR-31 inhibitor lentivirus group were significantly decreased (P < The relative surface area of myocardial cells decreased (P <0.05), while LATS2 increased slightly at gene level and protein level (P <0.05). Dual luciferase reporter assay showed that the relative luciferase activity of TRAF6-3’UTR + miR-146b was significantly lower than that of TRAF6-3’UTR + miR-NC (P <0.01), while LATS2-3’UTR + miR-31 The relative luciferase activity was significantly lower than that of LATS2-3’UTR-NC + miR-31 (P <0.01), and the differences were statistically significant. Conclusion: Down-regulation of miR-31 expression in hypertrophic cardiomyocytes can reverse the hypertrophy of cardiomyocytes to a certain extent. LATS2, a target of miR-31 targeting, regulates cardiomyocyte hypertrophy.