Abstract 004: Microrna-22 Regulates Smooth Muscle Cell Differentiation From Stem Cells By Targeting Methyl Cpg Binding Protein 2

Abstract

Objectives: To investigate the role of microRNA-22 (miR-22) in smooth muscle cell (SMC) differentiation from stem cells and the molecular mechanism involved. Methods and Results: Mouse embryonic stem (ES) cells were seeded on collagen IV-coated flasks and cultured in the absence of Leukemia Inhibitory Factor in differentiation medium for 4 to 8 days in order to induce SMCs differentiation. We found that miR-22 was significantly up-regulated during SMC differentiation. Enforced expression of miR-22 by its mimic in differentiating ES cells significantly up-regulated expression of a panel of SMC-specific genes, while knock-down of miR-22 by its antagomiR decreased these gene expression at both RNA and protein levels. Furthermore, we found over-expression and knockdown of miR-22 up-regulated and down-regulated SMC transcription factor SRF and its co-activator myocardin in a similar manner, respectively, and miR-22 overexpression in stem cells promoted SMC differentiation in vivo . Transcription repressor methyl CpG binding protein 2 (MECP2) was predicted as one of the top targets of miR-22 by using several computational miRNA target prediction tools. Interestingly, the gene expression levels of MECP2 were significantly decreased during SMC differentiation in a time dependent manner. Moreover, MECP2 was dramatically decreased in miR-22 overexpressing cells, but significantly increased when miR-22 was knockdown in the differentiating stem cells. Importantly, luciferase assay showed miR-22 substantially inhibited wild type MECP2-3’-UTR-luciferase activity in differentiating ES cells, but not mutant MECP2-3’-UTR-luciferease reporter. In addition, modulation of MECP2 expression levels affect multiple SMC-specific marker gene expression in differentiated ES cells by conducting gain-of-function experiments using MECP2 expression plasmid. Finally, we tested the expression of miR-22 in mouse femoral artery denudation injury model and found that miR-22 was significantly down-regulated in the wire injury-induced neointimal formation as early as day 3 post-injury. Conculsion: Using genomic and functional studies, we have discovered miR-22 controls SMC differentiation from stem cells via modulating MECP2 expression and function.