Figure 4.

ETS2 is a miR-146b-Regulated Transcription Factor Mediating MMP2 Upregulation in Human Bladder Cancer Cells

(A) Potential transcription factor-binding sites in the mmp2 promoter region (–1,459 to −42) were analyzed by using the TRANSFAC 8.3 engine online. (B and C) The indicated cell extracts were subjected to western blot for determination of the expression of c-Jun, JunB, Elk1, ETS2, and STAT5. GAPDH was used as a protein loading control. The results represented one of three independent experiments. (D) The overexpressed FLAG-ETS2 plasmid was stably transfected into T24T (miR-146b inhibitor) cells, and the cell extracts were then subjected to western blot for the determination of FLAG, ETS2, and MMP2 expressions, and GAPDH was used as a protein loading control. (E) The mmp2 promoter-driven luciferase reporter together with the TK reporter was transfected into the indicated cells. Luciferase activity of each transfectant was evaluated and the error bars show mean ± SD from three independent experiments. The asterisk (*) indicates a significant increase as compared with nonsense transfectant (p < 0.05). (F) ChIP assay was carried out using anti-ETS2 antibody to detect the interaction of ETS2 with the mmp2 promoter. (G and H) The invasion abilities of T24T (miR-146b inhibitor/vector) and T24T (miR-146b inhibitor/FLAG-ETS2) cells were determined, as described in the Materials and Methods. The error bars represent mean ± SD from three independent experiments. Student’s t test was utilized to determine the p value. The asterisk (*) indicates a significant increase in comparison to T24T (miR-146b inhibitor/vector) (*p < 0.05) (H).