miR-34a Inhibits HNSCC Tumor Growth Figure 7. miR-34a inhibits endothelial cell proliferation, migration and tube formation. A: miR-34a expression in endothelial cells expressing Bcl-2 (EC-Bcl-2) or vector alone (EC-VC) was analyzed by real time RT-PCR. B-C: Endothelial cells (EC) transfected with 50 nM of miR-34a or scrambled control RNA (SC) were used for different assays after 48 hrs of transfection. Endothelial cell proliferation (B) and cell migration (C) was measured using Xcelligence system. D: Endothelial cell tube formation assay was performed on Matrigel coated lab-tech chamber slides. At the end of assay, each chamber was photographed under microscope, and the area occupied by endothelial cell tubes was calculated using NIS-Elements-BS software (Nikon) and expressed as an angiogenic score (E). Whole cell lysates were Western blotted (72 hrs post transfection) and probed with specific antibodies against E2F3a/b, SIRT1, survivin and CDK4 proteins. Equal protein loading in Western blotting was verified by stripping the blots and reprobing with tubulin antibody. doi:10.1371/journal.pone.0037601.g007 analysis of the effect of miR-34a and E2F3 in HNSCC. In this study we show that miR-34a mediates its tumor suppressor effects predominantly through E2F3a isoform, as overexpression of miR resistant E2F3a significantly rescued the effects of miR-34a on cell proliferation and colony formation. Recently, Chen et al, demonstrated that out of two different E2F3 isoforms, E2F3a showed significantly higher oncogenic potential as compared to E2F3b [54]. Similarly, Reimer et al, have shown that although both E2F3 isoforms were overexpressed in tumor samples, but only E2F3a expression directly correlated with tumor stage and residual disease in ovarian cancer patients [55]. In addition, the same group has also shown that there was a strong correlation between E2F3a expression and activated EGFR in ovarian cancer specimens and that E2F3a was a key player in EGFR driven cell proliferation [56]. The EGFR pathway is abnormally activated in HNSCC and overexpression of EGFR has been associated with poor response to chemotherapy and poor survival in head and neck cancer patients [57]. Partial rescue of miR-34a function by E2F3b in our studies could be due to some functional overlap often observed in E2F3a and E2F3b [28,30]. Another important observation we made in this study is that ectopic expression of miR-34a in head and neck cancer cells also significantly down- regulated survivin expression. Recently, miR-34a was shown to modulate survivin promoter activity [34]. However, it is not known if miR-34a-mediated regulation of survivin promoter activity is due to its direct effect or mediated via another protein. Our rescue experiments with miR resistant E2F3a suggest that miR-34a may be inhibiting survivin expression via E2F3a [35]. We also observed an inverse correlation between miR-34a and survivin expression (low miR-34a and high survivin) in most of the PLoS ONE | www.plosone.org 9 May 2012 | Volume 7 | Issue 5 | e37601