miR-9-3p down-regulates E2F1 by directly targeting its 3′ UTR. (A) WT 3′ UTR of E2F1, E2F3, MDM2, or PDK1 or mutant 3′ UTR of E2F1 mRNA was cloned into pMIR-REPORT downstream of Firefly luciferase, and the resulting plasmid was designated pMIR-3′-UTR-E2F1, pMIR-3′-UTR-E2F3, pMIR-3′-UTR-MDM2, pMIR-3′-UTR-PDK1, or pMIR-3′-UTR-E2F1 mutant, respectively (the underlines represent the mutated seed regions). (B) Nontargeted or miR-9-3p mimic was transfected into MCF-7 cells together with pMIR-3′-UTR-E2F1, pMIR-3′-UTR-E2F3, pMIR-3′-UTR-MDM2, pMIR-3′-UTR-PDK1, or pMIR-3′-UTR-E2F1 mutant and a control Renilla luciferase expression vector. 60 h after transfection, cells were harvested and assayed for relative luciferase units. (C) MCF-7 cells were transfected with nontargeted miRNA, miR-9-3p mimic, or miR-9-3p antagomir. 60 h after transfection, cells were harvested, and E2F1 expression was analyzed using RT-qPCR at the mRNA level (left) or Western blotting at the protein level (right). (D, top) MCF-7 cells were treated with PP242 or rapamycin as indicated and analyzed for E2F1 expression as described in C. (Bottom) 36 h after transfection with a miR-9-3p antagomir gradient, MCF-7 cells were treated with 200-nM PP242 for an additional 24 h, and E2F1 expression was analyzed via Western blotting. Error bars represent mean values ± SEM. ns, not significant.