Figure 4

BCL2 and NTRK2 are direct targets of miR-204. Kelly cells were transfected with miR-204 mimics or scrambled negative control (scrambled NC) oligonucleotides. Graphed data are mean values ±s.e.m. of at least three independent experiments. (A) Conserved 8-mer seed match with hsa-miR-204 in BCL2 3′ UTR. (B) BCL2 mRNA relative expression to controls was determined by RQ-PCR at 24, 72 and 96 h post transfection, and BCL2 protein levels determined by western blot following total protein extraction at 48 h. (C) Kelly cells were cotransfected with miR-204 mimics or scrambled NC and either wild-type (WT) or mutated (mut) BCL2 3′-UTR reporter constructs. Luciferase activity was determined 48 h post transfection. (D) Conserved 8-mer seed match with hsa-miR-204 in NTRK2 3′ UTR. (E) Following transfection, Kelly cells were treated with 5 μ M ATRA for 48 h to induce full-length NTRK2 expression. NTRK2 mRNA expression relative to controls was determined by RQ-PCR at 72 and 96 h post transfection, and NTRK2 protein levels determined by western blot following total protein extraction at 72 h. (F) Kelly cells were cotransfected with miR-204 mimics or scrambled NC and either WT or mutant NTRK2 3′-UTR reporter constructs. Luciferase activity was determined 48 h post transfection.