摘要
目的:研究微小RNA-1(miR-1)对L-钙通道β2亚基的调控作用及对心肌细胞肥大的影响。方法:ISO诱导心肌细胞肥大。qRT-PCR和Western blot法分别检测心肌细胞ANP、β-MHC、miR-1和β2亚基mRNA和蛋白表达水平。应用Targetscan预测miR-1的靶基因。构建的重组质粒和miR-1共转染HEK293细胞。转染miR-1mimic使其过表达。RNAi干扰β2蛋白表达。激光共聚焦显微镜检测心肌细胞内钙离子浓度。结果:①ISO诱导心肌细胞肥大时,miR-1表达明显降低;转染miR-1mimic使其过表达,心肌细胞表面积、ANP和β-MHC mRNA表达均显著降低。②Targetscan预测显示,L-钙通道β2亚基为miR-1的潜在靶基因;将miR-1和含β23′UTR报告基因共转染HEK293细胞,其萤光值显著降低;转染miR-1mimic使其过表达,可明显抑制β2蛋白的表达。③ISO诱导心肌细胞肥大时,β2蛋白表达明显增加;RNAi干扰β2蛋白表达可明显抑制心肌细胞表面积、ANP和β-MHC mRNA表达的增加;④转染miR-1mimic使其过表达或RNAi干扰β2蛋白表达,心肌细胞内钙离子浓度均明显降低。结论:L-钙通道β2亚基为miR-1的靶基因。MiR-1可能通过负性调控L-钙通道β2亚基的表达,降低细胞内钙离子浓度,抑制心肌细胞肥大。
Objective：To investigate the effects of negative regulation of microRNA-1 （miR-1） on L-type calci- um channel β2 subunit during cardiac hypertrophy and its mechanism. Method：Cardiomyocyte hypertrophy was in- duced by ISO. The expression of ANP, β-MHC, miR-1 and β2 subunit mRNA were detected by qRT-PCR. The protein expression of β2 subunit was detected by Western blot. The targets of miR-1 were predicted by online Tar- getscan. The 3 untranslated region sequence of β2 was cloned into luciferase reporter vector and then transfected into HEK293 cells. The level of miR 1 was up-regulated by miR-1 mimic transfection. The expression level of β2 was down-regulated by RNAi. Intraeellular Ca2＋ concent was measured by confocal laser microscope. Result：① The expression of miR 1 was significantly reduced in cardiomyocyte hypertrophy. Upregulating the miR-1 level could suppress the increase of cell surface area, the expression of ANP and β-MHC mRNA. ②L-type calcium channel β2 subunit was the one of potential targets of miR-1 by prediction using online Targetscan. The luciferase activity of HEK293 cells with the plasmid containing widetype β2 3UTR sequence was significantly decreased when compared with control group. Upregulation of the miR-1 level could suppress the expression of β2 protein. ③The expression of β2 protein was significantly increased in cardiomyocyte hypertrophy. Downregulation of β2 expression by RNAi could markedly inhibit the increase of cell surface area , expression of ANP and β-MHC mRNA. ④Up- regulation of miR 1 level or downregulation of β2 expression markedly inhibited intracellcular Ca2＋ coneent. Conclu- sion：L-type calcium channel β2 subunit is truly the target of miR-1. MiR-1 could negtively regualte the expression of L-type calcium channel β2 subunit, resulting in the decrease of intracellular Ca2＋ concent and the attenuation of cardiomyocyte hypertrophy.
出处
《临床心血管病杂志》
CAS
CSCD
北大核心
2014年第6期546-550,共5页
Journal of Clinical Cardiology
