Abstract 834: Roles of Muscle-specific MicroRNAs During Differentiation of Mouse Embryonic Stem Cells into Cardiac Myocytes
Increasing evidence suggests important roles of microRNAs (miRs), short non-coding RNAs that repress translation or cleave RNA transcripts, in various biological processes. Previous studies report that miR-1 and miR-133 are involved in skeletal muscle differentiation, and that miR-143 is required for growth inhibition. However, the role of these miRs during differentiation of embryonic stem (ES) cells into cardiac myocytes is unknown. Mature miR-1, miR-133 and miR-143 were specifically amplified by real-time RT-PCR using miR-unique primers. These three miRs 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 miR normalized with that of 5S ribosomal RNA was increased. Among these three miRs, the expression level in the adult heart relative to that in undifferentiated ES cells was highest in miR-1 and lowest in miR-143. To examine the role of these miRs in myocardial differentiation, we overexpressed miR-1, miR-133, or miR-143 in ES cells by lentivirus-mediated gene transfer. Overexpression of miR-1 or miR-133 reduced the expression levels of cardiac-specific genes such as Nkx2.5 and ANF, while overexpression of miR-143 had no effect. The 3′ untranslated region (3′ UTR) of mRNAs for HDAC4 and cyclin-dependent kinase 9 (CDK9), possible targets of miR-1, were subcloned downstream of luciferase cDNA in the luciferase expression vector. The expression of miR-1 in ES cells reduced luciferase activity in the CDK9 –3′UTR construct, but not in the HDAC4 –3′UTR construct. Interestingly, trichostatin A (TSA), an inhibitor of histone deacetylase, markedly decreased the levels of muscle-specific miR-1 but not those of ubiquitously expressed miR-24, induced the amount of CDK9, 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 miR-1 represses the differentiation of ES cells into cardiac myocytes, in part, by de-stabilizing mRNA of CDK9.