Figure 5. ETV5 and TMPRSS2-ETV5 gene fusions prime E₂-ERβ2 mediated growth and migration of PC-3 cells through NF-κB dependent expression of cyclin D1 and MMPs in PC-3 cells.

(A) PC-3 cells were transiently transfected with siRNA targeting TMPRSS2 or non-targeting control siRNA control, and then treated with vehicle or 10 nM E₂ for 12 or 24 hours. Transcripts and protein levels of cyclin D1, TMPRSS2 and GAPDH were measured by RT-PCR (upper three panels) and immunoblot (lower three panels) analyses. (B) Transcript and protein levels cyclin D1, TMPRSS2 and ETV5 were examined by RT-PCR (upper three panels) and Western blot (lower three panels) in PC-3 cells transfected with siRNA targeting TMPRSS2 or non-targeting control siRNA, and then treated with vehicle or 10 nM E₂ for 12 or 24 hours, respectively. A transwell migration assay was performed to assess the number of PC-3 cells migration. The cells were transiently transfected with siRNA targeting TMPRSS2 or non-targeting control siRNA control and then treated with vehicle or E₂ (10 nM) and allowed to migrate to the bottom wells through a 4-μm pore membranes for 24 hours. (C–E). Migrated cells were stained with Calcein-AM uptake (C) or Diff-Quick method (D) or methylene blue (E, F) MMP2 and MMP9 gene expression in PC-3 cells transiently transfected with siRNA targeting TMPRSS2 or non-targeting control siRNA, and then treated with 10 nM E₂ or vehicle for 12 hours. (G) PC-3 cells transfected with siRNA targeting TMPRSS2 or non-targeting control siRNA, and then treated with vehicle or 10 nM E₂ for 24 hours. Cell lysates were subjected for immunoblot analysis with antibodies against NF-κB, pNF-κB (p65), pIkB, or GAPDH. Bar graphs represent mean ± SEM values in triplicates. * denotes significant difference compared to controls at P < 0.05 (n = 3).