Enhanced mesenchymal stem cell survival induced by GATA-4 overexpression is partially mediated by regulation of the miR-15 family.

We reported previously that pre-programming mesenchymal stem cells with the GATA-4 gene increases significantly cell survival in an ischemic environment. In this study, we tested whether regulation of microRNAs and their target proteins was associated with the cytoprotective effects of GATA-4. METHODS AND RESULTS: Mesenchymal stem cells were harvested from adult rat bone marrow and transduced with GATA-4 (MSC(GATA-4)) using the murine stem cell virus retroviral expression system. Cells transfected with empty vector (MSC(Null)) were used as controls. Quantitative real-time PCR data showed that the expression levels of miR-15 family members (miR-15b, miR-16, and miR-195) were significantly down-regulated in MSC(GATA-4). The protein expression of Bcl-w (Bcl-2-like-2), an anti-apoptotic Bcl-2 family protein, was increased in MSC(GATA-4). Hypoxic culture (low glucose and low oxygen) induced the release of lactate dehydrogenase from mesenchymal stem cells and reduced cell survival. Compared to MSC(Null), MSC(GATA-4) showed less lactate dehydrogenase release and greater cell survival following 72 h hypoxia exposure. The mitochondrial membrane potential, detected with the dye JC-1, was well maintained, and mitochondrial membrane permeability, expressed as caspase 3 and 7 activities in response to the ischemic environment was lower in MSC(GATA-4). Moreover, transfection with miR-195 significantly down-regulated Bcl-w expression in mesenchymal stem cells through a binding site in the 3'-UTR of the Bcl-w mRNA and reduced mesenchymal stem cell resistance to ischemic injury. CONCLUSIONS: The overexpression of GATA-4 in mesenchymal stem cells down-regulates miR-15 family members, causing increased resistance to ischemia through the up-regulation of anti-apoptotic proteins in the Bcl-2 family.