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 Takaya, Teruhisa Kawamura, Koh Ono, Rieko Takanabe, Shinji Kaichi, Tatsuya Morimoto, Hiromichi Wada, Toru Kita, Akira Shimatsu, Koji Hasegawa.

Division of Translational Research, National Hospital Organization Kyoto Medical Center, Kyoto, Japan.

The 25th Annual Meeting of the International Society of Heart Research Japanese Section (Yokohama, Japan), 2008/12/05 (Poster).

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 investigate the expression levels of miRNAs during differentiation of ES cells, mature miRNA-1, miRNA-133 and miRNA-143 were specifically amplified by real-time PCR using miRNA-unique primers. These miRNAs were strongly expressed in the adult mouse heart but very weakly expressed in undifferentiated mouse ES cells. Upon the induction of differentiation, the amount of each miRNA was increased. To examine the roles of miRNAs during cardiac differentiation, we overexpressed miRNA-1, miRNA-133, or miRNA-143 in ES cells by lentivirus-mediated gene transfer. 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 (UTR) of miRNA for cyclin-dependent kinase 9 (Cdk9) is 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 miRNA-1 but not those of ubiquitously expressed miRNA-24, and specifically enhanced myocardial differentiation. A dominant-negative form of Cdk9 inhibited the TSA-induced cardiac differentiation of ES cells, 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.

Keywords: microRNA, embryonic stem cells, cardiac differentiation.