with 39-untranslated regions (39-UTR) of specific target messenger RNA (mRNA) to suppress translation or occasionally induce their degradation [5]. Changes in miR expression profile have been shown to be associated with a variety of human cancers [8,9,10,11]. More importantly, it has been found that they are differentially expressed in tumor tissues compared to noncancerous tissues [12,13,14,15,16]. miRs can function as tumor suppressors or oncogenes, depending on whether they specifically target oncogenes or tumor suppressor genes. Therefore, understanding the molecular mechanisms by which these miRs play a role in deregulated cellular signaling in the head and neck cancer cell might help develop better therapeutic strategies for treatment of this disease. Recent studies have highlighted the role of miR-34a as a tumor suppressor in a number of tumor types including prostate cancer, hepatocellular carcinoma, neuroblastoma and colon cancer [17,18,19,20,21,22]. miR-34a was originally discovered as a potential tumor suppressor that is down- regulated and induces apoptosis in neuroblastoma cells [20]. Subsequently, it was shown to be a transcriptional target of p53 protein [23,24]. Functionally miR-34a was found to affect tumor cell proliferation, apoptosis, senescence, invasion, metas- tasis and drug resistance [21,22,23,25,26,27]. However, very little is known about the role and expression status of miR-34a in head and neck cancers. In this study, we examined if loss of miR-34a in head and neck cancers promotes tumor growth and tumor angiogenesis. Our results demonstrate that miR-34a expression is significantly downregulated in primary tumors from head and neck cancer patients as well as in head and neck cancer cell lines. Ectopic expression of miR-34a in head and neck cell lines significantly inhibited tumor cell proliferation, migration and colony formation by downregulating the expres- sion of E2F3 and survivin. In addition, miR-34a significantly inhibited tumor growth and tumor angiogenesis in a SCID mouse xenograft model. Interestingly, miR-34a inhibited tumor angiogenesis by blocking VEGF secretion by tumor cells as well as directly inhibiting endothelial cell functions. Results miR-34a Expression is Significantly Downregulated in Primary Tumor Samples and Tumor Cell Lines from the Head and Neck Cancer Patients We measured miR-34a expression levels in 30 frozen samples from head and neck cancer patients (15 tumors and 15 adjacent normal controls) by TaqMan real time RT-PCR. Out of 15 tumor samples, 6 were from oral cavity, 5 from oropharynx and 4 from larynx. Our results show that miR-34a expression is significantly decreased (Mann-Whitney test; p value 0.0226) in head and neck tumors as compared to adjacent normal tissue (Figure 1A). We did not observe any significant differences in miR-34a levels in tumor samples from the different head and neck sub-sites. We next examined if head and neck tumor cell lines also exhibit similar downregulated expression of miR-34a as compared to normal keratinocyes. We examined miR-34a levels in 3 different normal keratinocyte cell types [human oral keratinocytes (HOK); human epidermal keratinocytes, adult (HEKa) and human epidermal keratinocytes, neonatal (HEKn)] and 9 head and neck cancer cell lines. Indeed, miR-34a expression was markedly downregulated in all the head and neck cancer cell lines that were examined (Figure 1B). miR-34a Inhibits HNSCC Tumor Growth Figure 1. Expression of miR-34a is downregulated in HNSCC cell lines and patient’s tumor samples. A: miR-34a expression was examined in primary tumor samples (n = 15) or in adjacent normal mucosa (n = 15) of head and neck cancer patients by quantitative real time PCR (RT-PCR) and statistical significance was analyzed by Mann- Whitney test (p value 0.022). B: miR-34a expression was analyzed in 9 head and neck cancer cell lines by RT-PCR and compared with 3 normal human keratinocyes [human oral keratinocytes (HOK); human epider- mal keratinocytes, adult (HEKa) and human epidermal keratinocytes, neonatal (HEKn)]. doi:10.1371/journal.pone.0037601.g001 miR-34a Inhibits Head and Neck Tumor Cell Proliferation, Colony Formation and Migration Next, we examined the role of miR-34a on head and neck tumor cell proliferation, colony formation and migration. 50 nM of pre-miR-34a or scrambled control RNA was used to transfect head and neck cancer cell lines. Ectopic expression of miR-34a in transfected cells (UM-SCC-74A) was confirmed by RT-PCR (Figure 2A). Similar ectopic expression of miR-34a was observed in UM-SCC-74B cells (data not shown). We performed parallel experiments for cell proliferation using Xcelligence system and MTT assay (Roche Diagnostics, Indianapolis, IN). Ectopic expression of miR-34a significantly inhibited cell proliferation in UM-SCC-74A cells (MTT assay; Figure 2B & Figure S2 and Xcelligence assay; Figure 2C) and UM-SCC-74B cells (Figure 2D). Similarly, miR-34a transfection in both the head and neck cancer cell lines markedly decreased tumor cell colony formation (Figure 2E-H) and tumor cell migration (Scratch assay; Figure 3A and Xcelligence migration assay; Figure 3B-C). Cell migration by PLoS ONE | www.plosone.org 2 May 2012 | Volume 7 | Issue 5 | e37601