基础医学与临床
基礎醫學與臨床
기출의학여림상
Basic & Clinical Medicine
2015年
2期
196-202
,共7页
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
miR-1%miR-133a%L-型鈣通道β2 亞基%L-型鈣通道α1C亞基%心肌細胞肥大
miR-1%miR-133a%L-형개통도β2 아기%L-형개통도α1C아기%심기세포비대
miR-1%miR-133a%L-type calcium channelβ2 subunit%L-type calcium channelα1C subunit%cardiomyocyte hy-pertrophy
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microCosm 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-133 a 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,α1 C亚基是miR-133 a的靶基因。 miR-1和miR-133 a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。
目的:研究miR-1和miR-133a在大鼠心肌細胞肥大中對L-型鈣通道β2亞基( Cavβ2)和α1C亞基的調控作用。方法異丙腎上腺素( ISO )誘導大鼠心肌細胞肥大;在線數據庫microCosm 和Targetscan 預測miR-1和miR-133a的靶基因;分彆構建含有Cavβ23′UTR或α1C 3′UTR的重組質粒,與miR-1或miR-133a共轉染HEK293細胞,驗證Cavβ2亞基是miR-1的靶基因,α1C亞基是miR-133a的靶基因;用Western blot 法檢測心肌細胞內Cavβ2和α1C蛋白錶達;用siRNA榦擾Cavβ2和α1C錶達,明確Cavβ2和α1C在心肌細胞肥大中的作用。結果1) Cavβ2為miR-1的潛在靶基因,α1C為miR-133a的潛在靶基因。2)分彆將miR-1和Cavβ23′UTR,miR-133a和α1C 3′UTR共轉染HEK293細胞,熒光素酶熒光值均顯著降低( P<0.05, P<0.01)。3)分彆轉染miR-1 mimic、miR-133 a mimic上調miR-1、miR-133a的錶達後,心肌細胞內Cavβ2和α1C蛋白錶達均明顯下降(P<0.01, P<0.05)。4)用RNAi下調Cavβ2和α1C錶達可明顯抑製心肌細胞錶麵積(P<0.01),ANP和β-MHC mRNA錶達增加(P<0.05)。結論Cavβ2亞基是miR-1的靶基因,α1 C亞基是miR-133 a的靶基因。 miR-1和miR-133 a可能通過負性調控L-型鈣通道Cavβ2和α1C蛋白錶達,抑製心肌細胞肥大。
목적:연구miR-1화miR-133a재대서심기세포비대중대L-형개통도β2아기( Cavβ2)화α1C아기적조공작용。방법이병신상선소( ISO )유도대서심기세포비대;재선수거고microCosm 화Targetscan 예측miR-1화miR-133a적파기인;분별구건함유Cavβ23′UTR혹α1C 3′UTR적중조질립,여miR-1혹miR-133a공전염HEK293세포,험증Cavβ2아기시miR-1적파기인,α1C아기시miR-133a적파기인;용Western blot 법검측심기세포내Cavβ2화α1C단백표체;용siRNA간우Cavβ2화α1C표체,명학Cavβ2화α1C재심기세포비대중적작용。결과1) Cavβ2위miR-1적잠재파기인,α1C위miR-133a적잠재파기인。2)분별장miR-1화Cavβ23′UTR,miR-133a화α1C 3′UTR공전염HEK293세포,형광소매형광치균현저강저( P<0.05, P<0.01)。3)분별전염miR-1 mimic、miR-133 a mimic상조miR-1、miR-133a적표체후,심기세포내Cavβ2화α1C단백표체균명현하강(P<0.01, P<0.05)。4)용RNAi하조Cavβ2화α1C표체가명현억제심기세포표면적(P<0.01),ANP화β-MHC mRNA표체증가(P<0.05)。결론Cavβ2아기시miR-1적파기인,α1 C아기시miR-133 a적파기인。 miR-1화miR-133 a가능통과부성조공L-형개통도Cavβ2화α1C단백표체,억제심기세포비대。
Objective To investigate the regulation of miR-1 and miR-133 a on L-type calcium channel β2 subunit ( Cavβ2 ) and α1C subunit during rat cardiomyocyte hypertrophy .Methods Cardiomyocyte hypertrophy was in-duced by isoproterenol (ISO, 10μmol/L).The targets of miR-1 and miR-133a were predicted by online database microCosm and Targetscan , respectively .The 3′untranslated region sequences of Cavβ2 andα1C were respectively cloned into reporter vector and then transiently transfected into HEK 293 cells.The luciferase activities of samples were measured for demonstrating the expression of luciferase reporter vector .The protein expression of Cavβ2 andα1C were evaluated by Western blot .The expression levels of Cavβ2 andα1C were inhibited by RNAi to determine theeffectsofCavβ2andα1Concardiomyocytehypertrophy.Results 1)Cavβ2wasoneofpotentialtargetsof miR-1,α1C was the one of potential targets of miR-133a.2) The luciferase activities of HEK293 cells with the plasmid containing widetype Cavβ2 3′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 pro-tein expression of Cavβ2 and α1C, respectively(P<0.01, P<0.05).4)Downregulation of Cavβ2 andα1C by RNAi could markedly inhibit the increase of cell surface area ( P<0.01 ) , mRNA expression of ANP andβ-MHC (P<0.05).Conclusions Cavβ2 is the target gene of miR-1 and α1C is the target gene of miR-133a.miR-1 and miR-133a can negatively regulate the expression of L-type calcium channel Cavβ2 andα1C subunit, inhibi-ting cardiomyocyte hypertrophy.