miR-34a Inhibits HNSCC Tumor Growth Figure 2. Ectopic expression of miR-34a inhibits tumor cell proliferation and colony formation. A: Tumor cells (UM-SCC-74A) were transfected with miR-34a or scrambled control microRNAs (SC) and miR-34a expression was analyzed by quantitative real time PCR (RT-PCR). *, represent a significant difference (p,0.05). B: Cell proliferation was measured by MTT assay at different time points. Percentage cell proliferation for tumor cells transfected with miR-34a was calculated by adjusting proliferation index of tumor cells transfected with SC to 100. *, represent a significant inhibition (p,0.05) of cell proliferation in miR-34a expressing tumor cells as compared to SC. C-D: Cell proliferation was measured using Xcelligence system using the RTCA DP instrument. Percentage cell proliferation for tumor cells transfected with ectopic miR-34a was calculated by adjusting proliferation index of tumor cells transfected with SC to 100. *, represent a significant inhibition (p,0.05) of cell proliferation in miR-34a expressing tumor cells at 48 hrs as compared to SC. E-H: Effect of miR-34a on tumor cell colony formation (E-F; UM-SCC-74A and G-H; UM-SCC-74B) was examined by culturing tumor cells (5,000) in 60 mm Petri dishes for 14 days. Colony numbers in each assay was quantified by Alpha Innotech (San Leandro, CA) imaging software and percentage colony formation for tumor cells transfected with miR-34a was calculated by adjusting tumor cells transfected with SC to 100. *, represent a significant inhibition (p,0.05) of tumor cell colony formation in miR-34a expressing tumor cells as compared to SC. doi:10.1371/journal.pone.0037601.g002 PLoS ONE | www.plosone.org 4 May 2012 | Volume 7 | Issue 5 | e37601