tumor samples from HNSCC patients. Survivin is an important oncogene and its expression is directly associated with enhanced proliferation, resistance to chemotherapy [58,59], reduced apo- ptosis [60], enhanced angiogenesis [61], poor outcome [58,62] and increased rate of tumor recurrence [63]. These results provide a novel mechanistic role for the miR-34a-E2F3a-survivin axis in mediating miR-34a tumor suppressor function. The strong tumor suppressor effect of miR-34a on HNSCC cell lines observed during in vitro assays was further supported by its effects on in vivo xenograft tumor growth. In addition, miR-34a also significantly inhibited tumor angiogenesis by downregulating a key angiogenic factor VEGF. It has been shown that VEGF levels can be regulated by a number of miR-34a target proteins including E2F3, Myc and c-met [36,37,38]. Furthermore, our results demonstrate that miR-34a can also regulate tumor angiogenesis by directly inhibiting angiogenic functions of endothelial cells by downregulating a number of key proteins including E2F3, SIRT1, survivin and CDK4. Taken together, our results demonstrate an important tumor suppressor and anti- angiogenic function for miR-34a in head and neck cancers. Materials and Methods Ethics Statement This study was approved by the institutional review board at the Ohio State University and complied with all provisions of the Declaration of Helsinki. All animal work has been conducted according to the Ohio State University IACUC Animal ethic committee and was approved by this committee (Animal Welfare Assurance Number A3261-01). Patient Samples, Cell Lines and Reagents Tumor and adjacent normal tissue samples were collected from head and neck cancer patients undergoing surgical resection at the James Comprehensive Cancer Center at The Ohio State University. Use of these tissues was approved by the Ohio State University institutional review board. A board certified pathologist diagnosed all tumor tissue as HNSCC. Normal samples were collected from areas adjacent to the tumor tissue but outside the tumor margins. RNA was isolated from fresh frozen tissues samples using TRIzol reagent (Invitrogen). The isolated RNA was dissolved in RNase-free water and stored at 270uC. Out of the 15 tumor samples, 6 were from oral cavity, 5 from oropharynx and 4 from larynx. Head and neck squamous cell carcinoma (HNSCC) cell lines UM-SCC-74A, UM-SCC-74B, UM-SCC-47, UM-SCC- 49, UM-SCC-38 were obtained from Dr. Thomas E. Carey (University of Michigan). UD-SCC-2 was obtained from Dr. Henning Bier (Heinrich-Heine University, Germany). CAL 27, FaDu and SCC-25 were purchased from ATCC. The identity of all the tumor cell lines was confirmed by STR genotyping (Identifiler Kit, Applied Biosystems, Carlsband, CA) and cell line characteristics are presented in Table S1. Normal human oral keratinocytes (HOK) were purchased from ScienCell (Carlsbad, CA). Human epidermal keratinocytes, adult (HEKa) and human epidermal keratinocytes, neonatal (HEKn) were purchased from Invitrogen (Carlsbad, CA). All HNSCC cell lines, except SCC-25 were cultured in DMEM supplemented with 10% fetal bovine serum containing 1% penicillin/streptomycin (Invitrogen, Carls- bad, CA) and 1% Non-essential amino acids. SCC-25 was cultured in DMEM/F12 supplemented with 10% FBS, 1% penicillin/streptomycin, 400 ng/ml hydrocortisone. HOK, HEKa and HEKn were grown in keratinocyte growth medium (Invitro- gen, Carlsbad, CA). Primary human dermal microvascular endothelial cells (ECs) were purchased from Lonza (Walkersville, miR-34a Inhibits HNSCC Tumor Growth MD). ECs were maintained in Endothelial Cell Basal Medium-2 (EBM-2) containing 5% FBS and growth supplements. Primary antibody against E2F3a (clone 3E2F04) was purchased from Thermo Scientific (Fremont, CA), E2F3a/b (C-18) and CDK4 (C- 260) from Santa Cruz (Santa Cruz, CA), CD31 (clone MEC 13.3) from Diannova (Franklin Lakes, NJ) and survivin, SIRT1 and tubulin were purchased from Cell Signaling (Danvers, MA). Quantitative Reverse-transcription PCR (qRT-PCR) RNA was extracted from the HNSCC cell lines and normal keratinocytes (HOK, HEKa and HEKn) using the MirVANA kit (Ambion). TaqMan microRNA assay specific for miR-34a (Assay ID 000426) was used to detect and quantify mature miR-34a. For Survivin qRT-PCR, RNA was transcribed into cDNA and amplified with TaqMan primer/probe (Hs03043576_m1). The assays were performed in accordance with manufacturer’s instructions (Applied Biosystems, Carlsband, CA). miRNA and mRNA expression was normalized to RNU48 and OAZ1, respectively using the 2-DDCt method [64]. Ectopic Expression of miR-34a in HNSCC Cells and Endothelial Cells Precursor human miR-34a or scrambled control miRNA (Applied Biosystems) transfection in HNSCC tumor cells and EC was performed using Lipofectamine 2000 (Invitrogen) as per manufacturer’s instructions. In brief, HNSCC cells or endothelial cells were cultured in 6-well plates till they reached 60% confluence. Cells were washed and further cultured in the respective growth media minus antibiotics. In separate tubes, miR-34a (50 nM) and lipofectamine 2000 were diluted in OPTI- MEM medium and incubated for 5 minutes. After incubation, miR-34a and lipofectamine were mixed together and further incubated for 30 minutes. At the end of incubation, miR-34a and lipofectamine solution was carefully added to 6-well plates containing cells. After 24 hours, cells were washed and cultured in complete growth media containing antibiotics. To check transfection efficiency, FITC-labeled scrambled miRs were used and we consistently observed .80% transfection efficiency. Seventy two hours post transfection, cells were used for all the subsequent experiments. Transduction of Tumor Cells with E2F3a and E2F3b Retroviral particles were used to overexpress miR resistant E2F3a and E2F3b [29] (a kind gift from Dr. Brian D. Dynlacht, New York University) in head and neck cancer cell lines as described previously [65]. In brief, the E2F3a and E2F3b constructs or the vector alone was introduced into PT67 amphotropic packing cells with Lipofectamine 2000. Viral super- natants were collected after 24 h, centrifuged, filtered, and stored at 280oC. Head and neck tumor cells (UM-SCC-74A and UM- SCC-74B) were transduced with viral supernatants supplemented with 4 mg/ml polybrene 3 times every 4 hrs. The last infection was left overnight and the next morning cells were washed and cultured in fresh medium. Stable clones overexpressing E2F3a and E2F3b were selected by treating with puromycin (2 mg/ml) for 3 days. Cell Proliferation Tumor and endothelial cell proliferation were examined by MTT assay [66] (Roche Diagnostics, Indianapolis, IN) and Xcelligence system [67] using the RTCA DP instrument (Roche, Mannheim, Germany). For MTT assay, tumor or endothelial cells were plated in flat-bottomed 96-well microtitre plates at a density PLoS ONE | www.plosone.org 10 May 2012 | Volume 7 | Issue 5 | e37601