[Hsa-miR-133b expression profile during cardiac progenitor cell differentiation and its inhibitory effect on SRF expression]

ABSTRACT

Cardiac cell differentiation with the help of miRNAs has recently opened a promising window for the restoration of myocardial infarction. Independent miR-1-2/133a-1 and miR-206/133b clusters are known to be expressed in cardiac and skeletal muscles, respectively. miR-133b differs from miR-133a by only one nucleotide. The sequence similarity of these two miRNAs suggests that they target the same pathways and similar mRNA targets. The present study seeks to determine if miR-133b is expressed during the cardiac cell differentiation and if its expression is in reverse correlation with the SRF and CCND2 [as potential target genes] expression patterns. Human cardiac progenitor cells were prepared from Royan Stem Cell Bank [RSCB] and differentiated into cardiomyocytes. To initiate differentiation, cells were treated with 5-azacytidine as a demethylation factor. Then, ascorbic acid and TGFB1 were added every other day and twice per week, respectively. Differentiation into cardiomyocytes was confirmed by immunocytochemistry [ICC], flow cytometry and realtime PCR for some of the cardiac marker genes. The expression profiles of hsa-miR-133b and two of its potential target genes were also analyzed during the cardiac differentiation. Three weeks after the first differentiation induction, expression level of hsa-miR-133b was approximately five times higher than early stage expression [p<0.05]. During this process, the expression profile of SRF target gene was inversely correlated with hsamiR-133b expression. It is known that SRF is critically involved in the cell cycle. Considering increased miR-133b and decreased SRF expression levels during the late stages of heart cell differentiation, here we speculate that elevated expression of miR-133b blocks SRF expression and decreases cardiomyocytes proliferation in order to induce differentiation with direct targeting of SRF. Taken together, our data suggest that miR-133b along with miR-133a may be involved in cardiomyocytes differentiation.