Figure 2

MiR-204-5p is a negative modulator of TrkB expression in endometrial cancer cells. (A) Schema of selection of candidate miRNAs (miR-211-5p and miR-204-5p) targeting TrkB using three online target-prediction programs (TargetScan, Pictar and miRanda) and the microarray data. (B) Left panel: Schema of the construction of pMIRGLO-TrkB-3′UTR-WT and pMIRGLO-TrkB-3′UTR-MT vectors. The mutant binding site is underlined. Right panel: 293 T cells were co-transfected with pMIRGLO-TrkB-3′UTR-WT or pMIRGLO-TrkB-3′UTR-MT and miR-204-5p mimic (miR-204 m) or scrambled miRNA control (miR204m NC). Luciferase activities are shown as mean ± SD of at least three independent experiments done in triplicate. ** P < 0.01. (C) Ishikawa^TrkB cells were transfected with miR-204 m NC or miR-204 m. TrkB expression was examined by RT-PCR and normalized against β-actin. Data are shown as mean ± SD of at least three independent experiments.** P < 0.01; NS P > 0.05. (D) Cells were transfected as in (C) and TrkB expression was determined by Western blotting assays, which were quantified by densitometry of triplicate experiments (* P < 0.05; NS P > 0.05). β-actin was included as an internal control. Representative blots are shown in (E). (F) HEC-1B^shTrkB cells were transfected with a miR-204 inhibitor (miR-204i) or a scrambled control inhibitor (miR-204i NC) or with miR-204 inhibitor (miR-204i). TrkB expression was examined by RT-PCR and normalized against β-actin. Data are shown as mean ± SD of at least three independent experiments.** P < 0.01; NS P > 0.05. (G) Cells were transfected as in (F) and TrkB expression was determined by Western blotting assays, which were quantified by densitometry of triplicate experiments (** P < 0.01; NS P > 0.05). β-actin was included as an internal control. Representative blots are shown in (H).