Abstract 9169: Down-Regulation of miR-15 Family is Responsible for Overexpression of GATA-4 Mediated Cytoprotection
We have previously reported that mesenchymal stem cells (MSC) preprogrammed to express GATA-4 (MSCGATA-4) significantly increased their survival in ischemic myocardium. However, it is unclear how upregulation of GATA-4 in MSC mediates cytoprotection. MicroRNAs (miR) post-transcriptionally regulate the expression of thousands of distinct mRNAs. While some regulatory interactions help to maintain basal cellular functions, others are likely relevant in more specific settings, such as response to stress. In this study, we transduced GATA-4 into MSC. The expression of miR in MSC was assessed using miR microarray and quantitative real-time PCR. Bcl-2-like 2 (Bcl-w), one protein of miR-15 family targeted genes, was analyzed using qRT-PCR and western blot. Anoxia was used to mimic ischemic injury. FACS for annexin-V labeling cells was used to evaluate cell apoptosis and MTT intake was employed to study MSC survival. Our study indicated that overexpression of GATA-4 modulates the expression of several miR in MSC, including significantly decreased miR-15 family (e.g. miR-15b, miR-16, and miR-195) (Fig. 1). Bcl-2-like-2 (Bcl-w), which is one of target genes of miR-15 family and has an anti-apoptotic effect, was increased in MSCGATA-4. However, overexpression of miR-195 in MSCGATA-4 (MSCGATA-4-miR-195) using a lentiviral vector transfection system significantly reduced Bcl-w compared to that in the negative control miR transfected MSC (MSCGATA-4-miR-NC) (Fig. 2). The apoptosis of MSCGATA-4 following exposed to anoxia for 8 hours was lower than MSCNull, and it was abolished by transfecting miR-195 and siRNA-Bcl-w into MSCGATA-4 (Fig. 3). These results indicate that overexpression of GATA-4 downregulates miR-15 family resulting in upregulation of Bcl-w, responsible for GATA-4 mediated cytoprotection.
- © 2011 by American Heart Association, Inc.