摘要目的 研究微小RNA-133a(miR-133a)对L-型钙通道α1C亚基的调控作用及其对心肌细胞肥大的影响.方法 培养乳鼠心肌细胞,异丙肾上腺素(ISO)诱导心肌细胞肥大.相差显微镜和Leica图像分析软件测量心肌细胞表面积.逆转录实时定量PCR(qRT-PCR)和Western blot法分别检测心房钠尿肽(ANP)、β-肌球蛋白重链(β-MHC)、miR-133a和α1C mRNA和蛋白表达水平.转染miR-133a模拟物(mimic)上调心肌细胞miR-133a表达,观察对心肌细胞肥大的影响.应用网络数据库Targetscan预测miR-133a的靶基因.构建含α1C 3'UTR报告基因质粒和miR-133a瞬时共转染HEK293细胞,验证α1C为miR-133a靶基因.应用α1C RNAi干扰α1C蛋白表达,明确α1C亚基在心肌细胞肥大中的作用.应用激光共聚焦显微镜检测心肌细胞内钙离子浓度.结果 (1)在ISO诱导的心肌细胞肥大过程中,miR-133a表达显著下降(P＜0.01).miR-133a mimic转染心肌细胞上调miR-133a表达,心肌细胞表面积、ANP和β-MHC mRNA表达均明显降低(P均＜0.01).(2)网络数据库预测显示α1C为miR-133a的潜在靶点.将miR-133a和含α1C 3'UTR报告基因共转染HEK293细胞,其荧光值显著降低(P＜0.01).转染miR-133a mimic使心肌细胞miR-133a表达上调,可明显抑制α1C蛋白的表达(P＜0.05).(3)在ISO诱导心肌细胞肥大中α1C表达明显增加(P＜0.05).应用RNAi技术下调α1C表达可明显抑制心肌细胞表面积、ANP和β-MHC mRNA表达的增加(P分别＜0.01、0.05和0.05).(4)应用RNAi下调α1C表达或应用miR-133a mimic上调miR-133a表达,心肌细胞内钙离子浓度均明显降低(P均＜0.01).结论 α1C亚基为miR-133a的靶基因.miR-133a可能通过负性调控L-型钙通道α1C蛋白的表达,降低细胞内钙离子浓度、抑制心肌细胞肥大.

abstractsObjective To investigate the effects of microRNA-133a on isoproterenol (ISO)-induced neonatal rat cardiomyocyte hypertrophy and related molecular mechanism focusing on the changes of L-type calcium channel α1C subunit.Methods Neonatal rat cardiomyocytes were cultured,cardiomyocyte hypertrophy was induced by isoproterenol (ISO,10 μmol/L).The cell surface area was measured by phase contrast microscope and Leica image analysis system.The mRNA expressions of atrial natriuretic peptide (ANP),β-myosin heavy chain (β-MHC),miR-133a and the α1C were detected by qRT-PCR.The protein expression of α1 C was evaluated by Western blot.MiR-133a mimic was transfected into cardiomyocytes to investigate the effects of miR-133a on ISO-induced cardiomyocyte hypertrophy.The targets of miR-133a were predicted by online database Targetscan.The 3' untranslated region sequence of α1C was cloned into luciferase reporter vector and then transiently transfected into HEK293 cells.The luciferase activities of samples were measured to verify the expression of luciferase reporter vector.The expression level of α1C was inhibited by RNAi to determine the effects of α1 C on cardiomyocyte hypertrophy.Intracellular Ca2+ content was measured by confocal laser microscope.Results (1) The expression of miR-133a was significantly reduced in ISO-induced cardiomyocyte hypertrophy(P ＜ 0.01).Upregulating miR-133a level could suppress the increase of cell surface area,the mRNA expression of ANP and β-MHC (P ＜ 0.01).(2) α1C was the one of potential target of miR-133a by prediction using online database Targetscan.The luciferase activities of HEK293 cells with the plasmid containing widetype α1C 3' UTR sequence were significantly decreased compared with control group (P ＜ 0.01).Upregulation of the miR-133a level by miR-133a mimic transfection could suppress the protein expression of α1C (P ＜ 0.05).(3) The expression of α1C was significantly increased in ISO treated cardiomyocytes (P ＜ 0.05).Downregulation of α1 C by RNAi could markedly inhibit the increase of cell surface area,the mRNA expression of ANP and β-MHC(P ＜0.01,P ＜ 0.05,P ＜ 0.05).(4) Downregulation of α1 C expression by RNAi or upregulation of miR-133a level by miR-133a mimic transfection significantly inhibited intracellcular Ca2 + content (P ＜ 0.01).Conclusions Our data confirms that α1C is the target of miR-133a.MiR-133a can negatively regulate the expression of L-type calcium α1C subunit,resulting in the decrease of intracellular Ca2+ content and the attenuation of ISO-induced cardiomyocyte hypertrophy.