CSF), and found that these two agents mobilized different CD34 positive cell populations based on miR expression signatures, suggesting each miRs may regulate different group of BM cell mobilization [13–15]. However, the functional role of specific miRs and their targets for cell mobilization remains to be investigated. Here we report CXCR4 expression as a target of miR-150 which is downregulated in BM-derived MNCs in response to AMI, leading to MNC mobilization and migration in PB. Materials and Methods Experimental Mouse model of AMI C57BL/6 mice (female, 12 weeks old) were anesthetized by intrapertoneal injection of ketamine/xylazine (100 mg/kg and 20 mg/kg body weight, respectively). After endotracheal intuba- tion and mechanical ventilation using Harvard Appratus Mini- Vent (Type 845), a left intercostal thoracotomy was performed to open the chest. Left anterior descending coronary artery was permanently ligated with 6-0 silk (Echicon) and the chest was closed. For post-mortem studies, we sacrificed animals using overdose of sodium pentobarbital. All experimental procedures were performed in accordance with the standard human care guidelines of the ‘‘Guide for the Care and Use of Laboratory Animals’’ and were approved by the Institutional Animal Care and Use Committee of University of Cincinnati (Protocol ID: 06- 03-13-03), which conforms to National Institutes of Health guidelines. Isolation of MNCs One ml of peripheral blood was harvested from control and AMI mice by aspiration from heart. Total BM cells were obtained by flushing the cavity of femurs, tibias, and ilium and bone fragments were filtered through a 40 mm filter. MNCs were isolated by density-gradient centrifugation using 1.083 g/ml Histopaque 1083 solution (Sigma-Aldrich). Briefly, Histopaque 1083 was added to centrifuge tube and whole blood was carefully loaded onto surface of the histopaque. Then, histopaque with whole blood was centrifuged at 400 g for exactly 30 minutes at room temperature. After discarding upper layer, the opaque inferface containing the MNC layer was transferred into a 15 ml tube and washed with PBS twice by centrifugation. After the final wash, MNCs were used for further study. miRs profiling with microarray Total RNA samples obtained from PB- and BM-MNCs of control and AMI mice were sent to LC Sciences (Houston, TX) for miRs microarray profiling. Data were analyzed by LC Sciences with in-house developed computer programs. Intensity values were transformed into log2 scale, and fold changes were given in log2 scale. A t-test was performed between non-infarcted MNCs and infarcted MNCs from hearts, and statistical significance was considered at p,0.01. miRs isolation and detection Total RNA, including miRs, was extracted by using mirVana miR isolation kit (Ambion) in accordance with the manufacturer’s instructions. miR-150 was detected by using RT2 miRNA First Strand Kit (SA biosciences) and specific miR-150 primers from QIAGEN were used for quantitative reverse transcription (qRT) PCR and real time PCR. Relative expression was calculated using the comparative Ct method (22[D][D]Ct). miR-150 Targets Cxcr4 to Regulate MNC Mobilization Transfection of MNCs with miR inhibitor To knockdown miR-150 in MNCs, transfection was performed with anti-miR-150 (miRCURY LNA microRNA inhibitor, Exiqon) by electroporation system according to the instruction of manufacture (Electroporator II, Invitrogen). Briefly, 106106 BM- MNCs were resuspended in 400 ml of DMEM and transferred into electroporation cuvette. For miR inhibitor electroporation, 100 nM of anti-miR-150 was added and the loaded cuvettes were placed on ice. Voltage on the Electroporator II was set at 660 V and an infinite internal resistance value was used. A blank cuvette containing PBS were discharged at least twice before electropo- rating cells. MNCs were electroporated two times and then transferred to culture dish. The electroporated cells were incubated at 37uC in 5% CO2 and examined 48 hr after electroporation using an appropriate assay. Migration assay A total of 16106 BM-MNCs suspension in 250 ml serum-free EBM was placed in the upper chamber of Transwell Boyden chamber (Coster, 8 mm pore size, Sigma-Aldrich). Then the chamber was placed in 24 well culture dish containing 500 ml endothelial basal medium supplemented with 10% fetal bovine serum, singlequats and 100 ng/ml SDF-1 (Peprotech inc). After 24 hr of incubation at 37uC, the lower side of the filter was washed with PBS and fixed with 4% paraformaldehyde. Cells migrating to the lower chamber were stained with 0.1% crystal violet solution. Migration activity was evaluated as the mean number of migrated cells in three random microscopic fields (4006) per chamber. FACS analysis PB- and BM-derived MNCs were resuspended in PBS containing 1% bovine serum albumin and were incubated for 30 min in the dark at 4uC with phycoerythrin (PE) -conjugated monoclonal CXCR4 antibody (R&D). Cells were washed three times by centrifuging with 1 ml of PBS for 5 min at 400 g before analysis. Western blot analysis MNCs were lysed in lysis buffer, pH 7.4 [(in mM) 50 HEPES, 5 EDTA, 50 NaCl], 1% Triton X-100, protease inhibitors [(10 mg/ ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml leupeptin) and phosphatase inhibitors [(in mM) 50 sodium fluoride, 1 sodium orthovanadate, 10 sodium pyrophosphate]. The protein samples (40 mg) were electrophoresed using SDS- polyacrylamide gel and electro-immunoblotted as described. The specific antibody used for the detection of CXCR4 was purchased from ProSci Inc. miRs target gene prediction Computational analyses to predict potential binding between 39 UTR of target genes and miRNA was carried out with the following databases: microRNA.org (version 2010), TargetScan- Mouse (version 5.1) and MirBase (version 4). Only common targets were considered for experimental analysis. Lentivirus-mediated miR-150 inhibition in MNCs miR-150 overexpressing vector and scramble plasmid were obtained from GeneCopoeia. Lentivirus overexpressing miR-150 was generated by using Lenti-Pac HIV Expression Packaging Kit (GeneCopoeia). Briefly, 2.5 ml of lentiviral miR-150 inhibitor expression plasmid/control plasmid, 5.0 ml of EndoFectin Lenti and EndoFectin Lenti reagent were added in Opti-MEM I, and formed the DNA-EndoFectine complex. Twenty min after PLoS ONE | www.plosone.org 2 October 2011 | Volume 6 | Issue 10 | e23114