Abstract 14588: MicroRNA-34a Regulated Sirtuin 1 is Required for Smooth Muscle Cell Differentiation From Murine and Human Embryonic Stem Cells
Background: microRNA-34a (miR-34a) has been recently reported to regulate tumor cell cycle progression and apoptosis. However, very little is known about the functional role of miR-34a in smooth muscle cell (SMC) differentiation from stem cells. In the present study, we assessed the hypothesis that miR34a and its target genes play an important role in SMC differentiation.
Methods and results: Mouse embryonic stem (ES) cells were seeded on collagen IV-coated flasks and cultured in differentiation medium for 4 to 8 days to allow for SMC differentiation. miR-34a was significantly upregulated during SMC differentiation. Enforced expression and knockdown of miR34a in differentiating ES cells significantly promoted and inhibited SMC-specific gene expressions, respectively. Furthermore, overexpression and knockdown of miR-34a upregulated and downregulated several SMC transcription factors, including SRF, myocardin and MEF2c in a similar manner, and miR-34a overexpression in stem cells promoted SMC differentiation in vivo. Deacetylase sirtuin 1 (SirT1) was predicted as one of the top targets of miR-34a by using several computational miRNA target prediction tools. Surprisingly, SirT1 was positively regulated by miR-34a during SMC differentiation from stem cells. Luciferase assay showed miR-34a substantially activated wild type SirT1-3’-UTR-luciferase activity in differentiating ES cells, but not mutant SirT1-3’-UTR-luciferease reporter, further confirming a positive regulative role of miR-34a in SirT1 gene expression. Mechanistically, our data showed that differentiating stem cells underwent cell cycle arrest at G0/G1 phase, which was further promoted by miR-34 over-expression. Importantly, modulation of SirT1 expression levels affect multiple SMC-specific marker gene expressions in differentiated ES cells. Finally, miR-34a expression was also found to be significantly up-regulated during human ES cell differentiation, and miR-34a gene activation was required for SMC differentiation from human ES cells.
Conculsion: W e have uncovered an important role of miR-34a in SMC differentiation from murine and human ES cells, and successfully identified SirT1 as a functional modulating target in miR-34a mediated SMC differentiation.
- © 2013 by American Heart Association, Inc.