Diazoxide potentiates mesenchymal stem cell survival via NF-kappaB-dependent miR-146a expression by targeting Fas.
We have previously reported that preconditioning of bone marrow-derived mesenchymal stem cells (MSCs) with diazoxide (DZ) significantly improved cell survival via NF-κB signaling. Since micro-RNAs (miRNAs) are critical regulators of a wide variety of biological events, including apoptosis, proliferation, and differentiation, it is likely that DZ-induced survival is mediated by miRNAs. Here we show that miR-146a expressed during preconditioning with DZ is a key regulator of stem cell survival. Treatment of MSCs with DZ (200 μM) markedly increased miR-146a expression and promoted cell survival, as evaluated by lactate dehydrogenase release and transferase-mediated dUTP nick-end labeling staining. Interestingly, blocking NF-κB by IKK-γ NEMO binding domain inhibitor peptide did not induce miR-146a expression, indicating NF-κB regulates miR-146a expression. Moreover, blockade of miR-146a expression by antisense miR-146a inhibitor abolished DZ-induced cytoprotective effects, suggesting a critical role of miR-146a in MSC survival. Computational analysis found a consensus putative target site of miR-146a relevant to apoptosis in the 3' untranslated region of Fas mRNA. The role of Fas as a target gene was substantiated by abrogation of miR-146a, which markedly increased Fas protein expression. This was verified by luciferase reporter assay, which showed that forced expression of miR-146a downregulated Fas expression via targeting its 3'-UTR of this gene. Taken together, these data demonstrated that cytoprotection afforded by preconditioning of MSCs with DZ was regulated by miR-146a induction, which may be a novel therapeutic target in cardiac ischemic diseases.
Suzuki
Yoko
Y
Department of Pathology and Lab Medicine, University of Cincinnati, Cincinnati, OH 45267, USA.
Kim
Ha Won
HW
Ashraf
Muhammad
M
Haider
Husnain Kh
HKh
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003981:Diazoxide / Q000494:pharmacology*; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects*; D035683:MicroRNAs / Q000378:metabolism*; D023421:Models, Animal; D016328:NF-kappa B / Q000378:metabolism*; D051381:Rats; D011916:Rats, Inbred F344; D015398:Signal Transduction / Q000502:physiology; D033581:Stem Cell Transplantation; D019014:fas Receptor / Q000378:metabolism*
C491898:Fas protein, rat; D035683:MicroRNAs; D016328:NF-kappa B; D019014:fas Receptor; D003981:Diazoxide