Abstract 13086: Microrna-34a Regulates Smooth Muscle Cell Differentiation from Stem Cells by Targeting Sirtuin 1
Background: Smooth muscle cell (SMC) differentiation is a critical process during vascular development and disorders. MicroRNAs (miRNAs) have been reported to be involved in SMC differentiation. Recently, the microRNA-34a (miR-34a) was shown to regulate cell cycle progression and apoptosis. However, very little is known about the functional role of miR-34a in SMC differentiation. The aim of the present study is to dissect the role of miR34a during SMC differentiation from mouse embryonic stem (ES) cells and verify its targets.
Methods and results: We found that miR-34a was significantly upregulated during SMC differentiation by using real-time RT-PCR analysis. Furthermore, enforced expression of miR34a in differentiating ES cells was found to induce expression of all four SMC-specific genes (SMαA, SM22α, h1-calponin and SM-myh11). Similarly, Western blot analysis revealed the increased protein production of these genes in the cells overexpressing miR-34a. The knock-down of miR34a on the other hand, inhibited expression of these SMC-specific markers at both RNA and protein levels. Furthermore, we found over-expression and knockdown of miR-34a up-regulated and down-regulated several SMC transcription factors (SRF, myocardin and MEF2c) in a similar manner. Data from the microarray analysis revealed that sirtuin1 (SIRT1) was dramatically decreased in miR-34a overexpressing ES cells, which was further confirmed by real-time RT-PCR. Luciferase assay showed miR-34a substantially inhibited SIRT1-3’-UTR-luciferase activity in differentiating ES cells. Overexpression of miR34a was found to repress SIRT1 protein levels in cells, whereas miR-34a antagomir resulted in increased SIRT1 expression. In addition, we found that modulation of SIRT1 expression levels affect multiple SMC-specific marker gene expression in differentiated ES cells by conducting gain-of-function and lose-of-function experiments. Finally, we tested miR-34a expression in mouse femoral artery denudation injury model and found that miR-34a was significantly down-regulated as early as 3 days post-injury.
Conclusion: We have determined that miR-34a acts as an endogenous supressor of SIRT1 to control ES cell differentiation towards SMCs.
- © 2012 by American Heart Association, Inc.