Plasmid transfection and dual-luciferase reporter assay

293T cells were seeded on 96-well plates. miR-34c promoter-luciferase reporter plasmid was co-transfected with Renilla luciferase expression vector, pRL-TK (Promega), using Lipofectamine™ 2000 (Life Technologies), according to the manufacturers’ instruction. For co-transfection, pE2F1 was co-transfected with miR-34c promoter-luciferase reporter plasmid and pRL-TK. Twenty-four hours after transfection, the Firefly and Renilla luciferase activities were measured using Dual Luciferase Assay System (Promega) in Multiskan FC (Thermo Scientific), according to the manufacturers’ protocols. The ratio of Firefly/Renilla activity was calculated. All luciferase assays were performed in six times.