miR-34a Inhibits HNSCC Tumor Growth Figure 4. miR-34a mediates its biological function by downregulating E2F3 and survivin levels. A: UM-SCC-74A cells were transfected with miR-34a or scrambled control (SC). Seventy two hrs after transfection whole cell lysate was prepared and Western blotted using antibody specific for E2F3a (top panel) or antibody that binds both E2F3a and E2F3b (middle panel). Equal protein loading in Western blotting was verified by stripping the blots and reprobing with tubulin antibody. B: MicroRNA resistant E2F3a or E2F3b were stably overexpressed in UM-SCC-74A cells by retroviral vector and E2F3a and E2F3b overexpression was verified by Western blot analysis. C: UM-SCC-74A cells overexpressing E2F3a, E2F3b or vector alone (VC) were ectopically transfected with miR-34a or scrambled control (SC). After 72 hrs, cell proliferation was measured using Xcelligence system. D: Tumor cell colony formation was examined by culturing tumor cells (5,000) in 60 mm Petri dishes for 14 days and colony numbers is each assay was quantified by Alpha Innotech (San Leandro, CA) imaging software. E: UM-SCC-74A cells overexpressing E2F3a, E2F3b or vector alone (VC) were ectopically transfected with miR-34a or SC. After 72 hrs, whole cell lysates were Western blotted for survivin. Equal protein loading in Western blotting was verified by stripping the blots and reprobing with tubulin antibody. F: miR-34a, survivin and E2F3 mRNA expression from primary tumors of 15 head and neck cancer patients was analyzed by real time RT-PCR. doi:10.1371/journal.pone.0037601.g004 treatment. Therefore, it is imperative that new therapeutic strategies are developed to increase the long-term survival of these patients as well as decrease the adverse effects associated with concurrent chemo-radiation regimen. In order to develop tumor specific therapies, recent research efforts have attempted to exploit the biological differences that may exist between normal and malignant cells. Recent studies have demonstrated that microRNAs are differentially expressed in tumors as compared to normal tissues [8]. In our real-time PCR analysis of the head and neck cell lines, miR-34a expression was markedly downregulated in all the HNSCC cell lines that we tested. Similarly, Scapoli et al, have shown in their recent study that miR-34a levels are significantly downregulated in head and neck squamous cell carcinoma [44]. It is now being recognized that head and neck cancers can be subdivided into two distinct tumor types; HPV-negative and HPV- positive tumors and these tumor types have significantly different clinical outcome [45,46]. Recently, HPV infection was shown to down-regulate miR-34a levels by destabilizing tumor suppressor p53 protein in cervical cancer [47,48]. However, in our study, we did not observe any significant difference in miR-34a levels in HPV-positive as compared to HPV-negative head and neck cancer cell lines. In another study, Wald et al, also did not observe any significant difference in miR-34a levels in HPV-positive verses HPV-negative head and neck cancer cell lines [49]. This could be due to the fact that very few HPV positive head and neck cancer cell lines are available due to the difficulty in establishing HPV positive head and neck cell lines. In addition, the few HPV positive cell lines that are available might not represent the true nature of HPV positive head and neck tumors. We therefore selected two HNSCC cell lines (UM-SCC-74A and UM-SCC-74B) that are HPV negative and contain functional wild-type p53 for the in vitro and in vivo experiments, although they were not among the cell lines with the lowest miR-34a expression. The comparatively PLoS ONE | www.plosone.org 6 May 2012 | Volume 7 | Issue 5 | e37601