微小RNA-93负性调控信号转导与转录激活因子3通路逆转大鼠心肌肥厚的分子机制

摘要

Objective To investigate the role of microRNA (miRNA,miR)-93 in cardiac hypertrophy and its relation with signal transducer and activators of transcription 3 (STAT3) signal transduction pathway in vitro.And to identify the effect of miR-93 involved STAT3 pathway on cardiac hypertrophy cells.Methods The cultured neonatal rat cardiomyocyte was used to observe the hypertrophic effect of phenephrine (50 μmol/L) treatment for 48 h.After transfection with miR-93 (50 pmol/L),the hypertrophic response was assayed by measuring the cell diameter,protein content and miR-93 expression (50 pmol/L).The experiment of double luciferase report gene was used to elucidate interaction between miR-93 and STAT3.The expression of brain β-myosinheavychain (β-MHC),nuclear factor of activated T-cells (NFAT3),atrial natriuretic factor (ANF) and STAT3 pathway related protein was measured by Western blotting.Results In cultured cardiomyocytes,PE induced profound hypertrophic morphology change and the significant increase in cell diameter [(128 ± 28) μm],and protein content [(568.50 ± 61.24) pg/cell] in a concentration-dependent manner as compared with those in vehicle control (P ＜ 0.01).The contradictory result [(72 ±21) μm and (465.60 ±48.40) pg/cell,respectively] was found in cardiomyocytes transfected with miR-93 (P ＜0.01).miR-93 at concentration of 50 pmol/L significantly inhibited β-MHC,NFAT3,ANF and B cell lymphoma/leukemia-2 (bcl-2) and the protein expression of STAT3 as compared with those in the vehicle control (P ＜0.01).Interaction between miR-93 and STAT3 existed through uciferase report gene experiment.miR-93 markedly inhibited the myocyte hypertrophy (P ＜ 0.01).Conclusion Cardiomyocyte hypertrophy induced by PE may be,at least in part,mediated by STAT3 signal transduction pathway inhibited by increasing miR-93.Our results show that miR-93 may play a critical role as therapy factor in the cardiomyocyte hypertrophy.%目的 探讨大鼠心肌肥厚细胞中微小RNA(miRNA,miR)-93与信号转导与转录激活因子3(STAT3)的相关性,分析miR-93对STAT3调控的分子机制,观察miR-93通过STAT3通路对大鼠肥厚心肌细胞的影响.方法 应用酶消化法培养SD乳鼠心肌细胞,给予苯肾上腺素(PE,50 μmol/L)刺激48 h建立心肌肥厚模型,采用基因过表达转入miR-93(50 pmol/L),用苏木素-伊红(HE)染色观察心肌细胞形态学变化,同时检测细胞直径及蛋白含量,双荧光素酶报告基因实验检测miR-93与STAT3间相互作用,Western blot检测肌球蛋白链β(β-MHC)、活性T细胞核因子3(NFAT3)、心钠素(ANF)及STAT3通路相关蛋白.结果 转入miR-93组与PE对照组比较,细胞直径由(128±28) μm变为(72±21) μm(P＜0.01),蛋白含量由(568.50±61.24) pg/细胞变为(465.60±48.40) pg/细胞(P＜0.01),转入miR-93后,STAT3荧光素酶活性与对照组比较下降(P＜0.01),STAT3、β-MHC、NFAT3、ANF、B细胞淋巴瘤/白血病-2(bcl-2)蛋白均下调.结论 miR-93在大鼠心肌细胞中通过负性调控STAT3信号转导通路可能逆转心肌细胞肥厚.