TrkB represses miR-204-5p expression by activating the JAK2/STAT3 pathway. (A) TrkB inversely regulates miR-204-5p expression and (B) TRPM3 mRNA expression in endometrial cancer cells. MiR-204-5p and TRPM3 mRNA levels in Ishikawa and IshikawaTrkB cells and HEC-1B and HEC-1BshTrkB cells were measured by quantitative RT-PCR. MiR-204-5p expression was normalized against U6B and TRPM3 mRNA expression was normalized against β-actin. Data are expressed as mean ± SD of at least three independent experiments. **P < 0.01 and *P < 0.05. (C) TrkB, phospho-TrkB, phospho-P44/42, P44/42, JAK2, STAT3, phospho-JAK2 and phospho-STAT3 in Ishikawa and IshikawaTrkB cells and HEC-1B and HEC-1BshTrkB cells were assessed by Western blotting assays. Representative blots are shown. Blue arrows show the opposite changes in the two cell lines. (D) IshikawaTrkB and HEC-1B cells were transfected with siSTAT3, and phospho-STAT3 was assessed by Western blotting assays. (E) Inhibition of STAT3 increases miR-204-5p expression in IshikawaTrkB and HEC-1B cells. Data are expressed as mean ± SD of at least three independent experiments. *P < 0.05. (F) Schematic representation of the miR-204-5p locus and its host gene, TRPM3. STAT3-binding sites around the TRPM3 start site (UP1, DW1, and DW2) are predicted by in silico analysis. Chromatin immunoprecipitation assays were performed using anti-phospho-STAT3 antibody or rabbit isotype IgG. Immunoprecipitated DNA fragments were examined by quantitative real-time PCR (G). Data in (G) represent mean ± SD of at least three independent experiments performed in triplicate. VEGF was used as positive control and IgG as negative control.