Abstract 10704: microRNA-495 Mediates Cell Differentiation via DNA Methylation in P19 Stem Cells: An Endothelial Cell Model System
Background and Objective: The molecular basis for the relatively low efficiency of endothelial cell differentiation is not well understood. The present study uses microRNA-495 (miR-495) to promote the endothelial differentiation and investigate the mechanism in P19 embryonic carcinoma stem cells as a cell differentiation model system.
Methods: A miRNA expression profile was performed on original P19 cells after spontaneous differentiation (7-day) using a PCR array. Then miR-495 was overexpressed in P19 cells via lentivirus (P19miR-495, P19Null as control). The expression of endothelial cell marker genes (CD31 and CDH5) and pluripotent markers (Oct4 and Nanog) was analyzed by qPCR. The percentages of CD31+ and CDH5+ cells were assayed by flow cytometer. The methylation status of endothelial genes was analyzed by sodium bisulfite sequencing. DNMT3a, analyzed by luciferase reporter assay and qPCR, was predicted as a target of miR-495.
Results: Surprisingly we found that miR-495 expression was significantly increased during differentiation of original P19 cells (Fig. 1). The expression of miR-495, CD31 and CDH5 was gradually increased during a 14-day differentiation of original P19 cells cultured in endothelial differentiation medium, whereas Oct4 and Nanog expression was decreased. Compared to P19Null, the expression of CD31 and CDH5 was significantly increased in P19miR-495 after 14-day differentiation, while there was a significant decline in the percentage of methylated CG base sites of their promoter. The administration of a miR-495 analog decreased the luciferase activity of DNMT3a 3UTR reporter.
Conclusion: miR-495 represents a potential mediator for promoting endothelial differentiation, and thereby could guide more effective angiogenic treatments for myriad ischemic diseases.
- © 2013 by American Heart Association, Inc.