miR-34a mediates its biological function by downregulating E2F3 and survivin levels.

<p><i>A:</i> 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. <i>B:</i> 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. <i>C:</i> 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. <i>D:</i> 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. <i>E:</i> 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. <i>F:</i> miR-34a, survivin and E2F3 mRNA expression from primary tumors of 15 head and neck cancer patients was analyzed by real time RT-PCR.