miR-1和miR-133a对大鼠肥大心肌细胞L-型钙通道Cavβ_2和α1C亚基的调控作用  

Regulation of miR-1 and miR-133a on L-type calcium channel Cavβ_2 and α1C subunits in rat cardiomyocyte hypertrophy

作　　者：王玉琴[1] 耿鹏[2] 吴扬[2] 

机构地区：[1]南通大学附属医院神经内科,江苏南通226001 [2]南通大学航海医学研究所,江苏南通226001

出　　处：《基础医学与临床》2015年第2期196-202,共7页Basic and Clinical Medicine

基　　金：江苏省南通社会发展计划项目(S2010010)

摘　　要：目的研究miR-1和miR-133a在大鼠心肌细胞肥大中对L-型钙通道β2亚基(Cavβ2)和α1C亚基的调控作用。方法异丙肾上腺素(ISO)诱导大鼠心肌细胞肥大;在线数据库micro Cosm和Targetscan预测miR-1和miR-133a的靶基因;分别构建含有Cavβ23'UTR或α1C 3'UTR的重组质粒,与miR-1或miR-133a共转染HEK293细胞,验证Cavβ2亚基是miR-1的靶基因,α1C亚基是miR-133a的靶基因;用Western blot法检测心肌细胞内Cavβ2和α1C蛋白表达;用siRNA干扰Cavβ2和α1C表达,明确Cavβ2和α1C在心肌细胞肥大中的作用。结果 1)Cavβ2为miR-1的潜在靶基因,α1C为miR-133a的潜在靶基因。2)分别将miR-1和Cavβ23'UTR,miR-133a和α1C 3'UTR共转染HEK293细胞,荧光素酶荧光值均显著降低(P<0.05,P<0.01)。3)分别转染miR-1 mimic、miR-133a mimic上调miR-1、miR-133a的表达后,心肌细胞内Cavβ2和α1C蛋白表达均明显下降(P<0.01,P<0.05)。4)用RNAi下调Cavβ2和α1C表达可明显抑制心肌细胞表面积(P<0.01),ANP和β-MHC mRNA表达增加(P<0.05)。结论Cavβ2亚基是miR-1的靶基因,α1C亚基是miR-133a的靶基因。miR-1和miR-133a可能通过负性调控L-型钙通道Cavβ2和α1C蛋白表达,抑制心肌细胞肥大。Objective To investigate the regulation of miR-1 and miR-133 a on L-type calcium channel β2subunit（ Cavβ2） and α1C subunit during rat cardiomyocyte hypertrophy. Methods Cardiomyocyte hypertrophy was induced by isoproterenol（ ISO,10 μmol/L）. The targets of miR-1 and miR-133 a were predicted by online database micro Cosm and Targetscan,respectively. The 3＇ untranslated region sequences of Cavβ2and α1C were respectively cloned into reporter vector and then transiently transfected into HEK293 cells. The luciferase activities of samples were measured for demonstrating the expression of luciferase reporter vector. The protein expression of Cavβ2andα1C were evaluated by Western blot. The expression levels of Cavβ2and α1C were inhibited by RNAi to determinethe effects of Cavβ2and α1 C on cardiomyocyte hypertrophy. Results 1） Cavβ2was one of potential targets of miR-1,α1 C was the one of potential targets of miR-133 a. 2） The luciferase activities of HEK293 cells with the plasmid containing widetype Cavβ23 ＇ UTR sequence or α1 C significantly decreased（ P 〈0. 05,P 〈0. 01）.3） Upregulation of the miR-1 and miR-133 a by miR-1 mimic and miR-133 a mimic transfection suppressed protein expression of Cavβ2and α1 C,respectively（ P 〈0. 01,P 〈0. 05）. 4） Downregulation of Cavβ2and α1 C by RNAi could markedly inhibit the increase of cell surface area（ P 0. 01）,mRNA expression of ANP and β-MHC（ P 〈0. 05）. Conclusions Cavβ2is the target gene of miR-1 and α1 C is the target gene of miR-133 a. miR-1and miR-133 a can negatively regulate the expression of L-type calcium channel Cavβ2and α1 C subunit,inhibiting cardiomyocyte hypertrophy.

关 键 词：MIR-1 miR-133a L-型钙通道β2亚基 L-型钙通道α1C亚基 心肌细胞肥大 

分 类 号：R542.2[医药卫生—心血管疾病]