<p>(A) ERβ siRNA knockdown blocks fulvestrant-induced upregulation of <i>hsa-miR-765</i> expression in DU145 cells. Expression levels of <i>hsa-miR-765</i> determined by qRT-PCR analysis of the fulvestrant-treated cells with ERβ-siRNA (siERβ) or scramble negative-control (siNeg) were compared (n = 3). (B) SiRNA knockdown of ERβ blocks fulvestrant-induced transactivation of the 5′ upstream regulatory region of <i>hsa-miR-765</i> in DU145 cells. 5′ upstream regulatory region of <i>hsa-miR-765</i> was cloned into a luciferase vector. The reporter activities with ERβ-knockdown (siERβ) or scramble negative-control (siNeg) in the presence of fulvestrant were compared (n = 3). (C) Deletion mapping analysis defines a fulvestrant-responsive segment in <i>hsa-miR-765</i> regulatory region in DU145 cells. The 5′ upstream DNA sequence of <i>hsa-miR-765</i> from nt. −3208 to +100 was analyzed using luciferase reporter system. Serial deletions from the 5′ end of the cloned sequence in the vector were conducted. Reporter activities were compared between the fulvestrant-treated (Fulvestrant) and control (ETOH) cells for each reporter vector (n = 3). (D) Fulvestrant-induces recruitment of ERβ onto the putative <i>hsa-miR-765</i> regulatory region. Chromatin-immunoprecipitation revealed the recruitment of ERβ to a sequence in the 5′-regulatory region of <i>hsa-miR765</i>. Mouse IgG and RNA polymerase II serve as negative and positive control, respectively. Fulvestrant induced 17 fold increase in ERβ recruitment when compared with non-ERβ binding region (the 0N promoter of ERβ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098037#pone.0098037-Zhu1" target="_blank">[33]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098037#pone.0098037-Fernandes1" target="_blank">[41]</a>). Student's t-test was performed to determine significance of between groups using a cutoff p value of 0.05. ** p<0.01; bar = S.D.</p
