MicroRNA-1在心肌肥大中对L-型钙通道β_2亚基的负性调控作用  

Effects of microRNA-1 on negatively regulating L-type calcium channel β_2 subunit gene expression during cardiac hypertrophy

作　　者：吴扬[1] 耿鹏[1] 王玉琴[1] 刘艳[1] 

机构地区：[1]南通大学航海医学研究所,江苏南通226001

出　　处：《中国应用生理学杂志》2012年第4期304-308,共5页Chinese Journal of Applied Physiology

基　　金：江苏省南通市社会发展计划项目(S2010010)

摘　　要：目的:研究微小RNA-1(microRNA-1,miR-1)在心肌细胞肥大中对L-型钙通道β2亚基(Cavβ2)的负性调控作用及机制。方法:应用异丙肾上腺素(ISO)诱导心肌细胞肥大;采用HJ2000通用图像分析系统测定心肌细胞表面积;应用数据库microCosm预测miR-1的靶基因;构建含Cavβ23’UTR报告基因质粒和miR-1瞬时共转染HEK293细胞,验证Cavβ2为miR-1靶基因;应用qRT-PCR或Western blot方法检测心房钠尿肽(ANP)、β-肌球蛋白重链(β-MHC)、miR-1和Cavβ2mRNA和蛋白表达水平;转染miR-1模拟物上调miR-1或应用Cavβ2RNAi干扰Cavβ2蛋白的表达,观察对心肌细胞肥大的影响。结果:①在ISO诱导的心肌细胞肥大中,miR-1表达显著下降;应用miR-1 mimic转染心肌细胞使miR-1表达上调,心肌细胞表面积、ANP和β-MHC mRNA表达均显著低于ISO组(P<0.05)。②网络数据库预测显示Cavβ2为miR-1的潜在靶点;将miR-1和含Cavβ23’UTR报告基因质粒共转染HEK293细胞,其萤光值显著降低(P<0.01)。转染miR-1 mimic使心肌细胞miR-1表达上调,可以明显抑制Cavβ2蛋白的表达。③在ISO诱导心肌细胞肥大中Cavβ2表达较对照组显著增加;应用RNAi技术下调Cavβ2表达可明显抑制心肌细胞表面积、ANP和β-MHC mRNA表达的增加。结论:预测并验证L-型钙通道β2亚基为miR-1的靶基因。miR-1可能通过抑制其靶基因Cavβ2蛋白的表达,降低细胞内钙离子浓度,抑制心肌细胞肥大。Objective： To investigate the negative regulation of microRNA-1 （miR-1） on L-type calcium channel β2 subunit（Cavβ2） during eardiomyocyte hypertrophy and its mechanism. Methods： Cardiomyocyte hypertrephy was induced by isoproterenol （ISO）. The cell surface area was measured by image analysis system （HJ2000）. The targets of miR-1 were predicted by online database microcosm. The 3＇ untranslated region sequence of Cavβ2 was cloned into luciferase reporter vector and then transiently transfected into HEK293 cells. The lucifemse activities of samples were measured to verify the expression of lnciferase reporter vector. The expression of atrial natriuretic peptide（ANP） , β- myosin heavy chain（β-MHC）, miR-1 and the Cavβ2 mRNA were detected by qRT-PCR. The protein expression of Cavβ was detected by Western blot. The level of miR-1 was up-regulated by miR-1 mimic transfection and the expression level of Cavβ was down-regulated by RNAi, then effects of which on eardiomyecyte hypertrophy were investigated. Resutlts： （1）The expression of miR-1 was significantly reduced in cardiomyocyte hypertrophy. Upregulating the mill-1 level could suppress the increase of cell surface area, the expression of ANP and β-MHC mRNA（ P 〈0.05）. （2）Cavβ2 was the one of potential targets of miR-1 by prediction using online database microcosm. The lncifera. activites of HEK293 cells with the plasmid containing mill-1 and widetype Cave2 3＇ UTR sequence was significantly decreased when compared with that of control group（ P 〈 0.01 ）. Up-regulation of the miR-1 level could suppress the protein expression of Cave. （3）The expression of Cave2 was significantly increased in eardiomyocyte hypertrophy induced by ISO. Downregulation of Cavβ2 by PdNAi could markedly inhibit the of cell surface area, the expression of ANP and β-MHC mRNA. β- Cave2 is one of potential targets of miR-1 by bioinformatics prediction. The experiment data confirms that Cavβ is truly the target of miR- 1. Mill-1 c

关 键 词：MICRORNA-1 L-型钙通道β2亚基 心肌细胞肥大 

分 类 号：R331[医药卫生—人体生理学]