MicroRNA-133a attenuates isoproterenol-induced neonatal rat cardiomyocyte hypertrophy by downregulating L-type calcium channel α1C subunit gene expression
10.3760/cma.j.issn.0253-3758.2013.06.015
- VernacularTitle:微小RNA-133a可通过调控L-型钙通道α1C亚基的表达抑制乳鼠心肌细胞肥大
- Author:
Yang WU
1
;
Yu-Qin WANG
;
Bao-Xia WANG
Author Information
1. 226001,南通大学航海医学研究所
- Keywords:
Cardiomegaly;
RNA;
Calcium Channels,L-type
- From:
Chinese Journal of Cardiology
2013;41(6):507-513
- CountryChina
- Language:Chinese
-
Abstract:
Objective 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.
