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. 2014 May 16;9(5):e98037. doi: 10.1371/journal.pone.0098037

Figure 4. ERβ is involved in fulvestrant-induced upregulation of hsa-miR-765 expression.

Figure 4

(A) ERβ siRNA knockdown blocks fulvestrant-induced upregulation of hsa-miR-765 expression in DU145 cells. Expression levels of hsa-miR-765 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 hsa-miR-765 in DU145 cells. 5′ upstream regulatory region of hsa-miR-765 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 hsa-miR-765 regulatory region in DU145 cells. The 5′ upstream DNA sequence of hsa-miR-765 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 hsa-miR-765 regulatory region. Chromatin-immunoprecipitation revealed the recruitment of ERβ to a sequence in the 5′-regulatory region of hsa-miR765. 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 [33], [41]). 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.