miR-150 Targets Cxcr4 to Regulate MNC Mobilization Figure 2. Microarray-based miRs expression profiling in BM-MNCs. (A) Gene tree represented on left and heat map on right of 15 experiments, respectively (n = 5). The color of the top bar shows the degree of expression (red; upregulated genes, green; downregulated genes). (B) The list of miRs whose expression was altered in BM-MNCs in response to LAD ligation. doi:10.1371/journal.pone.0023114.g002 BM transplantation model Recipient C57BL/6 mice were exposed to a single sublethal dose of 5.5 Gy total-body c-irradiation. BM-MNCs from donor mice were infected with Lentivirus overexpressing miR-150 as described above. Lentivirus transfected-MNCs were counted, and 56106 cells were injected into recipient mice via retro-orbital venous sinus at 6–8 hr after irradiation. Injection into the retro- orbital sinus was easier to perform but more invasive than using the tail vein [16,17]. The mice were subjected to AMI and harvested MNCs from BM and PB at 5 days after AMI were analyzed as described above. Statistical analysis All values were expressed as means 6 SE. Comparison between two mean values was made by an unpaired Student two-tailed t- test. Statistical significance was accepted at p,0.05. Results AMI increases mobilization of BM-derived MNCs and CXCR4 positive cells into PB To examine whether AMI induces MNC mobilization from BM into PB, we assessed the number of MNCs in PB from mice at 1, 3 and 5 days after AMI and control mice as shown in Fig. 1A. The numbers of MNCs in PB were significantly decreased at day 1 after AMI (38.6% decrease) and markedly increased at day 3 (47.0% increase) and day 5 (110.7% increase) after AMI (Figure 1B). Furthermore, the percentage of CXCR4 positive MNCs isolated from both PB and BM of AMI mice was significantly increased (2.2 and 2.6 fold, respectively) as compared to those of non-infarcted mice (Figure 1C and D), indicating AMI induces mobilization of MNCs as well as CXCR4+ cells into blood circulation. miRs expression profiling in BM-derived MNCs from AMI mice To examine the potential participation of miRs in mobilization of MNCs, we performed miR profiling in BM-derived MNCs by using miRs microarray. As shown in Figure 2A and B, specific sets of miRs expression decreases more than 2 folds in BM-MNCs from AMI mice at 3 days as compared to control mice. These include miR-1937a (23.6 fold), miR-494 (23.39 fold), miR-29c (23.19 fold), miR-150 (22.95 fold), miR-486 (22.37 fold), miR- 30c (22.35 fold) and miR-30b (22.14 fold). Interestingly, miR- 29c, miR-150, and miR-494 expression was further decreased more than 4 fold at 5 days (27.28, 24.75, and 24.27, respectively), which led us to further study the role of these miRNAs in MNC mobilization. miR-150 regulates MNC migration by targeting Cxcr4 Since miR microarray analysis revealed that miR-29c, miR-98/ let-7 family, miR-150, miR-195 and miR-494 expression was significantly downregulated in BM-derived MNCs, we extensively examined databases for predicted targets of these miRNAs involved in MNC mobilization. Computational target prediction analysis indicated that Cxcr4 is one of the consensus putative targets of miR-150 relevant to stem cell mobilization (Figure 3A). To verify microarray profiling results, we performed real time PCR and confirmed that miR-150 expression was markedly reduced in BM-derived MNCs from AMI mice (Figure 3B). As shown in Figure 3C, knockdown of miR-150 by anti-miR-150 markedly reduced CXCR4 protein expression in MNCs, indicat- ing CXCR4 is a putative target of miR-150 in MNCs. To investigate the role of miR-150 in migration of MNCs, we performed in vitro cell migration assay by using Boyden Chamber transwell system. MNCs isolated from BM of AMI mice exhibited the higher number of migrated cells in response to SDF-1a as compared to control MNCs (Figure 3D, upper). Interestingly, knockdown of miR-150 by transfection with anti-miR-150 significantly increased the number of migrated MNCs when compared to scramble transfection, suggesting miR-150 is critically involved in MNC migration (Figure 3D, bottom). Knockdown of miR-150 enhances mobilization of MNCs in vivo To further examine the involvement of miR-150 in stem cell mobilization in vivo, we performed mouse BM transplantation experiments by using irradiated mice and lentiviral miR-150 inhibitor. Lentivirus containing mCherry (red, reporter) was generated by co-transfection of 293Ta cells with Lentiviral vectors and miR-150 inhibitor or scramble (Figure 4A). We produced Lentivirus containing anti-miR-150 and miR-scramble and transduced to MNCs isolated from healthy mice. Lentivirus transduction efficiency was confirmed by red signal of mCherry under microscope. Figure 4B showed approximately .90% of MNCs expressed mCherry at 2 days of Lentivirus infection. In addition, knockdown of miR-150 in MNCs by lentiviral vector significantly increased CXCR4 expression (Figure 4C). Interest- ingly, we found that in vivo transplantation of MNCs lacking miR- 150 expression (anti-miR-150MNCs) into the irradiated wild type mice resulted in increased number of MNCs in PB released from BM as compared to that of MNCs transducing scramble (ScMNCs), indicating that miR-150 plays a critical role in MNC mobilization in BM through Cxcr4 regulation (Figure 4D). Discussion It has been reported that transplantation of BM-MNCs in patients with ischemic heart failure reduces major adverse cardiovascular events after MI and improved cardiac function [18,19]. Several experimental studies suggest that most likely, multiple distinct cell populations of the BM-MNC are capable of contributing to neovascularization and differentiation into cardiac myocytes. MNCs systematically mobilized from BM can home to the sites of ischemic tissues such as infarcted myocardium, ischemic hindlimb, and the mechanism of MNCs homing to ischemic tissues has been extensively investigated. miRs are critically involved in many biological processes in health and disease including cardiovascular diseases. Although biological relevance of miRs in many stem cell functions is reported, the involvement of miRs in stem cell mobilization and migration is unknown. In this study, we reported here for the first time that the expression of miR-150 was downregulated in BM MNCs in response to myocardial ischemia with simultaneous induction of CXCR4 protein expression. These molecular events led to PLoS ONE | www.plosone.org 5 October 2011 | Volume 6 | Issue 10 | e23114