Fig. 4: AR induces ZIC5 expression via miR-27b-3p downregulation.
From: AR-regulated ZIC5 contributes to the aggressiveness of prostate cancer
A LNCaP cells were cultured in charcoal-stripped serum medium for 3 days and then administered various doses of dihydrotestosterone (DHT) for 48 h. ZIC5 protein levels were detected using western blot. B Western blot analysis of AR and ZIC5 expression in 22RV1 cells treated with DHT (1 nmol/L) to activate AR and in C4-2B cells transfected with AR-specific siRNA (si-AR). C Western blot analysis of AR and ZIC5 expression in 22RV1 and C4-2B cells treated with or without DHT (1 nmol/L) and ENZ (10 μmol/L). D, E RT-qPCR analysis of relative ZIC5 mRNA levels in 22RV1 cells (D) treated with DHT (1 nmol/L) and in C4-2B cells (E) transfected with si-AR. N.S., P > 0.05 vs. NC, *P < 0.05, vs. NC. F 22RV1 cells treated with DHT (1 nmol/L) and C4-2B cells transfected with si-AR were immunoprecipitated with an Ago2 antibody, and relative ZIC5 mRNA levels in the Ago2 complex were detected by RT-qPCR. *P < 0.05, vs. NC. G Western blot analysis of ZIC5 expression following transfection of 22RV1 cells with sh-ZIC5, miR-27b-3p inhibitors, and miR-27b-3p mimics. H Potential miRNA-27b-3p binding sites on the ZIC5 3′-UTR. I, J Luciferase activities were determined in 22RV1 cells cotransfected with wild or mutant ZIC5 3′UTR vectors and miR-27b-3p inhibitors, and in C4-2B cells cotransfected with wild or mutant ZIC5 3′UTR vector and miR-27b-3p mimics. *P < 0.05, vs. NC, N.S., P > 0.05 vs. NC. K Western blot analysis of ZIC5 expression in 22RV1 cells treated with or without DHT and transfected with miR-27b-3p mimics, and in C4-2B cells cotransfected with or without si-AR and miR-27b-3p inhibitors.
