[微小RNA-199a对大鼠心肌肥大的影响]
[Effect of miRNA-199a on rat cardiac hypertrophy].
作者信息
Xu Xu-dong, Song Xiao-wei, Jing Qing, Qin Yong-wen
机构信息
Department of Cardiology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China.
出版信息
Zhonghua Xin Xue Guan Bing Za Zhi. 2011 May;39(5):446-50.
OBJECTIVE
To investigate the role of miRNA-199a on cardiac hypertrophy.
METHODS
(1) Male Sprague-Dawley rats were subjected to pressure overload induced by abdominal aortic constriction (AAC, n = 6) and quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the change of microRNAs (miRNAs). (2) Neonatal rat ventricular myocytes were isolated from 2-day old Sprague-Dawley rats. The myocytes were divided into two groups: adenovirus miRNA-199a (Ad-miRNA-199a) or adenovirus vector (Ad-vector). They were transfected in cardiomyocytes for 48 h using Lipofectamine 2000. qRT-PCR was used to detect the change of myocardial hypertrophy markers α-myosin heavy chain (αMHC, myh6), β-myosin heavy chain (βMHC, myh7) and atrial natriuretic peptide (ANP, Nppa). Software Axio Vision was used to detect the change of cardiomyocytes surface areas. (3) Neonatal rat ventricular myocytes were divided into two groups: antisense oligonucleotide-miRNA-199a (As-miRNA-199a) and scramble oligonucleotides (As-ctl). They were transfected to cardiomyocytes respectively for 48 h. qRT-PCR was used to detect the change of miRNA-199a. (4) Neonatal rat ventricular myocytes were divided into four groups: A: control (ctl), B: phenylephrine (PE), C: PE + As-ctl, D: PE + As-miRNA-199a. qRT-PCR was used to detect the change of myh6, myh7 and Nppa. Software Axio Vision was used to detect the change of cardiomyocytes surface areas.
RESULTS
(1) qRT-PCR results showed that miRNA-1, miRNA-133, miRNA-181a and miRNA-499 were significantly decreased, while the miRNA-199a was significantly increased at 1 week post AAC hearts compared with the sham group. (2) qRT-PCR results showed that miRNA-199a and myh7 were increased and myh6 was decreased significantly in Ad-miRNA-199a group compared with Ad-vector group. The cardiomyocytes surface area was increased in Ad-miRNA-199a group detected by immunofluorescence. (3) qRT-PCR results showed that miRNA-199a was significantly decreased in As-miRNA-199a group compared with Ad-vector group. (4) The Nppa and myh7 were significantly increased and myh6 was decreased in cardiomyocytes stimulated by PE for 48 h. The cardiomyocytes surface area determined by immunofluorescence was increased in PE + As-miRNA-199a groups compared with PE + As-ctl groups.
CONCLUSION
miRNA-199a may play a regulatory role in cardiac hypertrophy.
目的
探讨miRNA - 199a在心肌肥厚中的作用。
方法
（1）对雄性Sprague - Dawley大鼠进行腹主动脉缩窄（AAC）诱导压力超负荷（n = 6），采用定量实时聚合酶链反应（qRT - PCR）检测微小RNA（miRNAs）的变化。（2）从2日龄Sprague - Dawley大鼠分离新生大鼠心室肌细胞。将肌细胞分为两组：腺病毒miRNA - 199a（Ad - miRNA - 199a）组和腺病毒载体（Ad - vector）组。使用Lipofectamine 2000将它们转染到心肌细胞中48小时。采用qRT - PCR检测心肌肥厚标志物α - 肌球蛋白重链（αMHC，myh6）、β - 肌球蛋白重链（βMHC，myh7）和心钠素（ANP，Nppa）的变化。使用Axio Vision软件检测心肌细胞表面积的变化。（3）将新生大鼠心室肌细胞分为两组：反义寡核苷酸 - miRNA - 199a（As - miRNA - 199a）组和乱序寡核苷酸（As - ctl）组。分别将它们转染到心肌细胞中48小时。采用qRT - PCR检测miRNA - 199a的变化。（4）将新生大鼠心室肌细胞分为四组：A：对照组（ctl），B：去甲肾上腺素（PE）组，C：PE + As - ctl组，D：PE + As - miRNA - 199a组。采用qRT - PCR检测myh6、myh7和Nppa的变化。使用Axio Vision软件检测心肌细胞表面积的变化。
结果
（1）qRT - PCR结果显示，与假手术组相比，AAC术后1周心脏中miRNA - 1、miRNA - 133、miRNA - 181a和miRNA - 499显著降低，而miRNA - 199a显著升高。（2）qRT - PCR结果显示，与Ad - vector组相比，Ad - miRNA - 199a组中miRNA - 199a和myh7升高，myh6显著降低。免疫荧光检测显示Ad - miRNA - 199a组心肌细胞表面积增加。（3）qRT - PCR结果显示与Ad - vector组相比，As - miRNA - 199a组中miRNA - 199a显著降低。（4）PE刺激48小时的心肌细胞中Nppa和myh7显著升高，myh6降低。免疫荧光检测显示，与PE + As - ctl组相比，PE + As - miRNA - 199a组心肌细胞表面积增加。
结论
miRNA - 199a可能在心肌肥厚中发挥调节作用。
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