Overexpression of microRNA-1 in mouse embryonic stem cells represses myocardial differentiation

Overexpression of microRNA-1 in mouse embryonic stem cells represses myocardial differentiation

Tomohide Takaya1, Teruhisa Kawamura1, Koh Ono2, Rieko Takanabe1, Shinji Kaichi3, Toru Kita2, Hiromichi Wada1, Tatsuya Morimoto1, Akira Shimatsu1, Koji Hasegawa1.

  1. Division of Translational Research, Kyoto Medical Center, National Hospital Organization, Kyoto, Japan.
  2. Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
  3. Department of Pediatrics, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

第72回日本循環器学会学術集会 (福岡), 2008/03/29 (口演).

Abstract

MicroRNAs (miRNAs), short non-coding RNAs that repress translation or cleave RNA transcripts, are involved in various biological processes. Previous studies report that miRNA-1 and miRNA-133 are involved in skeletal muscle differentiation, and that miRNA-143 is required for growth inhibition. However, the role of these miRNAs during differentiation of embryonic stem (ES) cells into cardiac myocytes is unknown. To solve this problem, we overexpressed miRNA-1, miRNA-133, or miRNA-143 in ES cells by lentivirus-mediated gene transfer. Efficient overexpression was confirmed by real-time RT-PCR using miRNA-unique primers. Overexpression of miRNA-1 or miRNA-133 reduced the expression levels of cardiac-specific genes such as Nkx2.5 and ANF, while overexpression of miRNA-143 had no effect. The 3' untranslated region (3' UTR) of mRNA for cyclin-dependent kinase-9 (Cdk9) is a possible target of miRNA-1. Overexpression of miRNA-1 in ES cells reduced the luciferase activity from the luciferase cDNA with Cdk9-3' UTR construct. Interestingly, trichostatin A (TSA), an inhibitor of histone deacetylase, markedly decreased the levels of muscle-specific miRNA-1 but not those of ubiquitously expressed miRNA-24, and specifically enhanced myocardial differentiation. A dominant-negative form of Cdk9 inhibited TSA-induced differentiation of ES cells into cardiac myocytes, suggesting a role of Cdk9 in myocardial differentiation. These findings demonstrate that miRNA-1 represses the differentiation of ES cells into cardiac myocytes, in part, by de-stabilizing mRNA of Cdk9.