increased by AMI and identified miR-150 as a key regulator of BM-MNC mobilization by targeting Cxcr4.. Knocking down miR- 150 by transfection with its inhibitor in MNCs exhibited the higher response to SDF-1a in migration capacity as compared with scramble-transfected MNCs. Furthermore, lentivirus-medi- ated knockdown of miR-150 significantly increased CXCR4 expression in MNCs and transplantation of these cells into BM of wild type mice dramatically increased MNC mobilization from BM to PB, indicating that miR-150 is critically associated with stem cell mobilization by regulating Cxcr4. miR-150 is abundantly expressed in monocytes [22] which is altered under various conditions including the immune response and tumorigenesis [23,24]. Therefore, miR-150 is being studied for its participation in addressing different aspects of cellular functions with special focus on its role in mobilization of monocytes. In this regard, c-Myb, a transcription factor related to endothelial cell migration and B cell differentiation, is being tipped as a potential target gene of miR-150 [22,25]. Microvesicle- based exogenous miR-150 delivery to human microvascular endothelial cells resulted in downregulation of c-MYB expression and enhanced their migration ability in vitro [25]. Although these data support our findings to imply miR-150 in cell mobilization, the discrepancy between the two studies may be attributed to; 1) different cell types (endothelial cell vs stem cell), 2) cell maturity (mature vs progenitor cell), 3) exogenous vs endogenous expression of miR-150, 4) disease type (atherosclerosis vs myocardial References 1. Shintani S, Murohara T, Ikeda H, Ueno T, Sasaki K, et al. (2001) Augmentation of postnatal neovascularization with autologous bone marrow transplantation. Circulation 103(6): 897–903. 2. Kamihata H, Matsubara H, Nishiue T, Fujiyama S, Tsutsumi Y, et al. (2001) Implantation of bone marrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands, and cytokines. Cirulationc 104(9): 1046–52. 3. Strauer BE, Brehm M, Zeus T, Ko¨stering M, Hernandez A, et al. (2002) Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circulation 106(15): 1913–8. 4. Jackson KA, Majka SM, Wang H, Pocius J, Hartley CJ, et al. (2001) Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Invest 107: 1395–1402. 5. Kocher AA, Schuster MD, Szabolcs MJ, Takuma S, Burkhoff D, et al. (2001) Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med 7: 430–436. 6. Dignat-George F, Sampol J (2000) Circulating endothelial cells in vascular disorders: new insights into an old concept. Eur J Haematol 65: 215–220. 7. Peled A, Petit I, Kollet O, Magid M, Ponomaryov T, et al. (1999) Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4. Science 283(5403): 845–8. 8. Yin Y, Zhao X, Fang Y, Yu S, Zhao J, et al. (2010) SDF-1alpha involved in mobilization and recruitment of endothelial progenitor cells after arterial injury in mice. Cardiovasc Pathol 19(4): 218–27. 9. Tang YL, Zhu W, Cheng M, Chen L, Zhang J, et al. (2009) Qin G. Hypoxic preconditioning enhances the benefit of cardiac progenitor cell therapy for treatment of myocardial infarction by inducing CXCR4 expression. Circ Res 104(10): 1209–16. 10. Haider HKh, Jiang S, Idris NM, Ashraf M (2008) IGF-1-overexpressing mesenchymal stem cells accelerate bone marrow stem cell mobilization via paracrine activation of SDF-1alpha/CXCR4 signaling to promote myocardial repair. Circ Res 103(11): 1300–8. 11. Winter J, Jung S, Keller S, Gregory RI (2009) Diederichs S. Many roads to maturity: microRNA biogenesis pathways and their regulation. Nat Cell Biol 11: 228–234. 12. Kim VN, Han J, Siomi MC (2009) Biogenesis of small RNAs in animals. Nat Rev Mol Cell Biol 10: 126–139. 13. Donahue RE, Jin P, Bonifacino AC, Metzger ME, Ren J, et al. (2009) Plerixafor (AMD3100) and granulocyte colony-stimulating factor (G-CSF) mobilize different CD34+ cell populations based on global gene and microRNA expression signatures. Blood 114(12): 2530–41. miR-150 Targets Cxcr4 to Regulate MNC Mobilization ischemia), all of which might have led to the different results between these studies. Another research group has identified a molecular cascade (PLZF transcription factor/miR-146a/ CXCR4) controlling normal megakaryopoiesis which involved enhanced expression of PLZF and inhibition of miR-146a transcription [26]. They demonstrated downregulation of miR- 146a elevated the translation of the target CXCR4 mRNA. In this regulatory loop, miR-146a acted as an effector of PLZF and mediated its control activity on Cxcr4 translation. Taken together, these studies indicated the functional significance of miR-150 in immune system and disease control and, warrant further investigations to elucidate the functional role of miR-150 in angiomyogenesis mediated by mobilized MNCs. In conclusion, our results demonstrated that miR-150 down- regulation by AMI enhanced CXCR4 expression, leading to enhanced BM-MNCs mobilization and migration. Thus, miR-150 may be a novel therapeutic target for stimulating BM cell mobilization to ischemic tissue for participation in the repair process. Author Contributions Conceived and designed the experiments: NT HWK MA. Performed the experiments: NT. Analyzed the data: NT HWK. Contributed reagents/ materials/analysis tools: HWK MA. Wrote the paper: NT HWK MA. 14. Georgantas RW, 3rd, Hildreth R, Morisot S, Alder J, Liu CG, et al. (2007) CD34+ hematopoietic stem-progenitor cell microRNA expression and function: a circuit diagram of differentiation control. Proc Natl Acad Sci U S A 104(8): 2750–5. 15. Jin P, Wang E, Ren J, Childs R, Shin JW, et al. (2008) Differentiation of two types of mobilized peripheral blood stem cells by microRNA and cDNA expression analysis. J Transl Med 6: 39. 16. Lessner S, Prado HL, Waller EK, Galis ZS (2002) Atherosclerotic lesions grow through recruitment and proliferation of circulating monocytes in a murine model. Am J Path 160: 2145–55. 17. Duran-Struuck R, Dysko RC (2009) Principle of bone marrow transplantation (BMT): Providing optimal veterinary and husbandry care to irradiated mice in BMT studies. J Am Assoc Lab Anim Sci 48: 11–2. 18. Scha¨chinger V, Erbs S, Elsa¨sser A, Haberbosch W, Hambrecht R, et al. (2006) REPAIR-AMI Investigators. Improved clinical outcome after intracoronary administration of bone-marrow-derived progenitor cells in acute myocardial infarction: final 1-year results of the REPAIR-AMI trial. Eur Heart J 27(23): 2775–83. 19. Pokushalov E, Romanov A, Chernyavsky A, Larionov P, Terekhov I, et al. (2010) Efficiency of intramyocardial injections of autologous bone marrow mononuclear cells in patients with ischemic heart failure: a randomized study. J Cardiovasc Transl Res 3(2): 160–8. 20. Ceradini DJ, Kulkarni AR, Callaghan MJ, Tepper OM, Bastidas N, et al. (2004) Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1. Nat Med 10(8): 858–64. 21. Heeschen C, Lehmann R, Honold J, Assmus B, Aicher A, et al. (2004) Profoundly Reduced Neovascularization Capacity of Bone Marrow Mononu- clear Cells Derived From Patients With Chronic Ischemic Heart Disease. Circulation 109: 1615–1622. 22. Xiao C, Calado DP, Galler G, Thai TH, Patterson HC, et al. (2007) MiR-150 controls B cell differentiation by targeting the transcription factor c-Myb. Cell 131(1): 146–59. 23. Tsitsiou E, Lindsay MA (2009) microRNAs and the immune response. Curr Opin Pharmacol 9(4): 514–20. 24. Amaral FC, Torres N, Saggioro F, Neder L, Machado HR, et al. (2009) MicroRNAs differentially expressed in ACTH-secreting pituitary tumors. J Clin Endocrinol Metab 94(1): 320–3. 25. Zhang Y, Liu D, Chen X, Li J, Li L, et al. (2010) Secreted Monocytic miR-150 enhances targeted endothelial cell migration. Mol Cell 39(1): 133–44. 26. Labbaye C, Spinello I, Quaranta MT, Pelosi E, Pasquini L, et al. (2008) A three- step pathway comprising PLZF/miR-146a/CXCR4 controls megakaryopoiesis. Nat Cell Biol 10(7): 788–801. PLoS ONE | www.plosone.org 8 October 2011 | Volume 6 | Issue 10 | e23114