Rapid communicationExpression of microRNAs is dynamically regulated during cardiomyocyte hypertrophy
Introduction
MicroRNAs (miRNAs) are a class of short, non-coding RNA molecules that have recently emerged as important regulators of gene expression [1]. Many miRNAs are highly evolutionarily conserved and more than 400 miRNAs are known to exist in humans alone. miRNAs negatively regulate target gene expression through miRNA complementarity to target sequences within the 3′ untranslated region of target mRNAs through translational repression or by degradation of bound RNA [1], [2], [3]. Recently, we and others have demonstrated that a subset of muscle-specific miRNAs, miR-1 and miR-133 in particular, play important roles in muscle cell proliferation and differentiation [4], [5]. Given that miRNAs participate in such fundamental processes, we investigated the potential role for miRNAs in regulating the well-documented changes in gene expression that occur during cardiac hypertrophy.
In this report, we show that the expression of a subset of miRNAs is altered in hypertrophic cardiomyocytes. Interestingly, we found more miRNAs that are up-regulated than down-regulated in response to cardiac hypertrophy. We have used both overexpression and knockdown approaches to demonstrate that miR-18b and miR-21 repress hypertrophy in neonatal rat cardiomyocyte in vitro.
Section snippets
Cardiomyocyte tissue culture and transfection
Preparation of neonatal rat cardiomyocytes was as described [6], [7]. LNA oligonucleotides and miRNA duplexes were transfected using Lipofectamine. Hypertrophic stimuli used: PE (100 μM), LIF (1000 units/ml) and fetal bovine serum (FBS) 10%.
Analysis of microRNA expression by microarray
Total RNA was isolated from rat neonatal cardiomyocytes and mouse heart tissue (Trizol). Microarray production and hybridization methods have been described [8]. Five micrograms of labeled RNA from each treatment was hybridized to a microRNA microarray with
Global analysis of miRNA expression during cardiac hypertrophy
To better understand the role of miRNAs in cardiac hypertrophy, we sought to identify miRNAs involved in cardiac hypertrophy using a custom miRNA microarray screening approach [4], [8]. Using this sensitive and quantitative high-throughput technology, we profiled global miRNA expression in phenylephrine (PE)-treated neonatal cardiomyocytes (Fig. 1A) and in mouse hearts subjected to pressure overload by thoracic aortic banding (TAB) (Figs. 1B and F) and found that the expression signatures of
Acknowledgments
We thank Xiaoyun Hu for excellent technical assistance and Wang laboratory members for their support. M.T. is a postdoctoral fellow and T. E. C. is a predoctoral fellow of the AHA. This study is supported by the March of Dimes Foundation, Muscular Dystrophy Association and NIH.
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