Wu Yang, Wang Yu-qin, Wang Bao-xia
Institute of Navigation Medicine, Nantong 226001, China (Email:
Zhonghua Xin Xue Guan Bing Za Zhi. 2013 Jun;41(6):507-13.
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.
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 α1C 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 α1C on cardiomyocyte hypertrophy. Intracellular Ca(2+) content was measured by confocal laser microscope.
(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 wide type 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 α1C 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 α1C expression by RNAi or upregulation of miR-133a level by miR-133a mimic transfection significantly inhibited intracellular Ca(2+) content (P < 0.01) .
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 Ca(2+) content and the attenuation of ISO-induced cardiomyocyte hypertrophy.
探讨微小RNA-133a（miR-133a）对异丙肾上腺素（ISO）诱导的新生大鼠心肌细胞肥大的影响及相关分子机制，重点关注L型钙通道α1C亚基的变化。
培养新生大鼠心肌细胞，用异丙肾上腺素（ISO，10 μmol/L）诱导心肌细胞肥大。通过相差显微镜和徕卡图像分析系统测量细胞表面积。采用qRT-PCR检测心房钠尿肽（ANP）、β-肌球蛋白重链（β-MHC）、miR-133a和α1C的mRNA表达。通过蛋白质印迹法评估α1C的蛋白表达。将miR-133a模拟物转染到心肌细胞中，以研究miR-133a对ISO诱导的心肌细胞肥大的影响。通过在线数据库Targetscan预测miR-133a的靶标。将α1C的3'非翻译区序列克隆到荧光素酶报告载体中，然后瞬时转染到HEK293细胞中。测量样品的荧光素酶活性以验证荧光素酶报告载体的表达。通过RNA干扰抑制α1C的表达，以确定α1C对心肌细胞肥大的影响。用共聚焦激光显微镜测量细胞内Ca(2+)含量。
（1）在ISO诱导的心肌细胞肥大中，miR-133a的表达显著降低（P < 0.01）。上调miR-133a水平可抑制细胞表面积的增加、ANP和β-MHC的mRNA表达（P < 0.01）。（2）通过在线数据库Targetscan预测，α1C是miR-133a的潜在靶标之一。与对照组相比，含有野生型α1C 3'UTR序列质粒的HEK293细胞的荧光素酶活性显著降低（P < 0.01）。通过转染miR-133a模拟物上调miR-133a水平可抑制α1C的蛋白表达（P < 0.05）。（3）在ISO处理的心肌细胞中，α1C的表达显著增加（P < 0.05）。通过RNA干扰下调α1C可显著抑制细胞表面积的增加、ANP和β-MHC的mRNA表达（P < 0.01、P < 0.05、P < 0.05）。（4）通过RNA干扰下调α1C表达或通过转染miR-133a模拟物上调miR-133a水平可显著抑制细胞内Ca(2+)含量（P < 0.01）。
我们的数据证实α1C是miR-133a的靶标。miR-133a可负向调节L型钙通道α1C亚基的表达，导致细胞内Ca(2+)含量降低，减轻ISO诱导的心肌细胞肥大。
