Xcelligence was measured at 24 hrs and there was 58% and 63% cell migration inhibition in UMSCC-74A and UM-SCC-74B cells, respectively (Figure 3B-C). Whereas only 20% cell proliferation inhibition (Figure 2) was observed at the same time point (24 hrs), thereby suggesting that miR-34a predominantly affected cell migration at 24 hrs. miR-34a Inhibits Tumor Cell Proliferation and Colony Formation by Downregulating E2F3 and Survivin We next sought to identify the target mRNAs of miR-34a that regulate head and neck tumor cell function. TargetScan data base (www.targetscan.org) search revealed several growth regulatory mRNAs that contains conserved miR-34a recognition sites in their 39-UTR. In this study, we focused our attention to E2F3a and E2F3b because E2F3 family of transcription factors play an important role in cell cycle regulation, cell proliferation and differentiation [28,29,30]. To confirm that miR-34a regulates the endogenous expression of E2F3a and E2F3b, we ectopically introduced miR-34a in UM-SCC-74A cells and examined E2F3a/ b levels in these cells 72 hrs post transfection. We used two different antibodies against E2F3, including a monoclonal anti- body that specifically recognizes the unique N-terminal portion of E2F3a (Figure 4A-B, top panel) and this antibody is referred as E2F3a. We also used a polyclonal antibody that recognizes a region close to C terminus, which is common to both E2F3a and E2F3b [29] (Figure 4A-B, middle panel). Expression of both E2F3a and E2F3b proteins was markedly downregulated in miR-34a transfected cells as compared to cells transfected with scrambled control RNA (Figure 4A). As both E2F3 isoforms were downregulated in miR-34a transfected cells, we performed isoform-specific rescue experi- ments to understand the specific contribution of E2F3a and E2F3b in miR-34a mediated tumor suppressor function. We used microRNA resistant constructs for E2F3a and E2F3b [29]. These constructs were generated by modifying the microRNA targeting sequence by introducing three silent base changes (a gift from Dr. David Dynlacht, New York University). Expression of E2F3a and E2F3b in UM-SCC-74A cells showed significantly elevated protein levels as compared to cells transfected with control vector (VC) (Figure 4B). Overexpression of E2F3a or E2F3b in UM- SCC-74A cells showed a modest increase in cell proliferation (12% and 6% respectively; Figure 4C). Interestingly, E2F3a over- expression in UM-SCC-74A cells significantly rescued tumor cells from miR-34a-mediated inhibition of cell proliferation (56%, Figure 4C) and colony formation (86%, Figure 4D), whereas E2F3b was only partially effective in reversing miR-34a-mediated inhibition of cell proliferation (19%, Figure 4C) and colony formation (26%, Figure 4D). Survivin, encoded by the gene BIRC5 is a member of the inhibitor of apoptosis proteins (IAP) family of molecules [31]. Survivin is aberrantly expressed in many malignancies including HNSCC [32] and has been shown to play a role in cancer progression and resistance to therapy [33]. Recently, miR-34a was shown to decrease survivin promoter activity [34]. In addition, survivin promoter activity is also regulated by E2F3, a key miR- 34a target protein [35]. Since ectopic expression of miR34a inhibits E2F3 expression, we further examined if survivin expression is regulated by miR-34a via E2F3. Indeed, miR-34a transfection in UM-SCC-74A cells significant decreased survivin protein levels (Figure 4E). We next examined if overexpression of miR resistant E2F3a or E2F3b could rescue survivin expression in miR-34a treated cells. Overexpression of E2F3a was able to completely rescue survivin expression in miR-34a transfected UM- SCC-74A cells, whereas E2F3b was only partially effective miR-34a Inhibits HNSCC Tumor Growth (Figure 4E). To further validate the cell line findings of inverse relationship between miR-34a and its target proteins survivin and E2F3, we examined miR-34a, survivin and E2F3 mRNA levels in the primary head and neck tumor samples. All 15 tumor samples showed an inverse relationship between miR-34a levels and survivin (Pearson r value 0.89; p value 0.0001). 14/15 patient samples showed low levels of miR-34a and high levels of survivin mRNA. One sample (Patient # 10) showed high miR-34a and low survivin levels (Figure 4F). Similarly, miR-34a levels were inversely correlated with E2F3 levels (Pearson r value 0.81; p value 0.0002). miR-34a Inhibits Tumor Growth, in vivo To confirm the anti-tumor effects on miR-34a in vivo, we implanted two head and neck cell lines either expressing miR-34a or scrambled control in the right or left flanks of SCID mice, respectively. Representative photographs of SCID mice bearing tumors at day 18 and dissected tumors are shown in Figure 5C-F. Ectopic expression of miR-34a expression in both the tumor cell lines significantly inhibited tumor growth in SCID mice (Figure 5). In order to confirm that ectopic expression of miR-34a stayed throughout the in vivo experiments, we isolated RNA from UM- SCC-74A tumor samples at the end of study and performed real time RT-PCR. Indeed, ectopic expression of miR-34a was maintained in the tumor cells throughout the study (Figure 5G). miR-34a Inhibits Tumor Angiogenesis by Downregulating VEGF Secretion from Tumor Cells In our in vivo tumor growth study, in addition to smaller tumor size, we also observed that tumors were visually less vascular in miR-34a transduced group. VEGF is a key angiogenic protein and recent studies have shown that a number of miR-34a target proteins including E2F3, Myc and c-met can regulate VEGF expression [36,37,38]. Therefore, we next examined if ectopic expression of miR-34a affected tumor angiogenesis. UM-SCC- 74A tumors expressing miR-34a showed a significant decrease in blood vessel density (83%) as compared to tumors expressing scrambled control RNA (Figure 6A-B). Similarly, UM-SCC-74B tumors showed 87% decrease in tumor blood vessel density (Figure 6D-E). We next examined if miR-34a inhibits tumor angiogenesis by blocking VEGF production by the tumor cells. miR-34a was ectopically expressed in tumor cells and VEGF (VEGF A) levels in the culture supernatants was quantified by ELISA. Untreated UM-SCC-74A cells produced very high levels of VEGF (1247 pg/ml/106cells) and miR-34a transduction significantly reduced VEGF production (56%) (Figure 6C). Sim- ilarly, miR-34a transfection in UM-SCC-74B cells significantly reduced VEGF production (45%) (Figure 6F). miR-34a Inhibits Endothelial Cell Proliferation, Migration and Tube Formation In our previous study, we have shown Bcl-2 expression is significantly elevated in tumor-associated endothelial cells (EC- Bcl-2) [39,40] and our results from this study suggest that miR-34a expression is significantly decreased in Bcl-2 expressing endothelial cells as compared to endothelial cells containing empty vector alone (EC-VC) (Figure 7A). We next examined if miR-34a could directly affect angiogenic function of endothelial cells in vitro. Transfection of miR-34a in endothelial cells significantly inhibited cell proliferation (85%) and migration (84%) (Figure 7B-C). Similarly, miR-34a significantly inhibited the ability of endothelial cells to form tubular structure on Matrigel (Figure 7Da-b). In addition, ectopic expression of miR-34a in endothelial cells PLoS ONE | www.plosone.org 3 May 2012 | Volume 7 | Issue 5 | e37601