miR-34a Inhibits HNSCC Tumor Growth Figure 6. miR-34a inhibits tumor angiogenesis by downregulating VEGF secretion from tumor cells. A-B: Paraffin embedded tumor samples were stained with anti-CD31 antibody and microvessel density was calculated by counting 5 random high power fields (200x). A: Representative photographs of UM-SCC-74A tumors expressing scrambled control microRNAs (SC) or miR-34a. B: Microvessel density in tumor samples from scrambled control microRNAs (SC) or miR-34a groups. *, represent a significant inhibition (p,0.05) of tumor angiogenesis in miR-34a group as compared to SC. C: VEGF levels (pg/ml per 106 tumor cells) in culture supernatants from UM-SCC-74A cells transfected with miR-34a or scrambled control microRNA (SC). D: Representative photographs of UM-SCC-74B tumors expressing scrambled control microRNAs (SC) or miR-34a. E: Microvessel density in tumor samples from scrambled control microRNAs (SC) or miR-34a groups. F: VEGF levels (pg/ml per 106 tumor cells) in culture supernatants from UM-SCC-74B cells transfected with miR-34a or scrambled control microRNA (SC). doi:10.1371/journal.pone.0037601.g006 higher miR-34a levels in these cell lines could most likely be due to the presence of wild-type p53 in these cells as miR-34a has been shown to be a direct transcriptional target of p53 [23,24]. UM- SCC-74B cell line is derived from an intraoral head and neck tumor and it is a relatively sensitive cell line to chemotherapy and radiation treatment. In contrast, UM-SCC-74A cell line is derived from the base of the tongue tumor and is highly resistant to both chemotherapy and radiation treatment [50]. Similar to HNSCC cell lines, miR-34a expression was also significantly downregulated in tumor samples from head and neck cancer patients. This remarkable decrease in miR-34a in head and neck tumors suggested to us that its deregulation in HNSCC may be playing a role in the progression of head and neck cancer. To test this hypothesis, we performed a series of experiment using in vitro as well as in vivo models. Our results demonstrate that ectopic expression of miR-34a strongly suppresses multiple tumorigenic functions (e.g. proliferation, and colony formation) of head and neck cancer cells. In addition, ectopic expression of miR-34a significantly inhibited cell migration even after taking into account the effect of miR-34a on cell proliferation at the same time point (60% cell migration inhibition as compared to 20% cell proliferation inhibition at 24 hrs). These tumor suppressive effects of miR-34a are mediated by changes in a number of target mRNAs including MYCN, CDK4, cyclin D1, SIRT1 and Bcl-2 [18,51,52,53]. In this study, we looked at changes in protein expression of some of the known targets of mir-34a (e.g. SIRT1, CDK4, E2F3) and found them to be altered upon ectopic miR- 34a expression (Figure S1). We decided to conduct an in-depth PLoS ONE | www.plosone.org 8 May 2012 | Volume 7 | Issue 5 | e37601