<i>Hsa-miR-765</i> suppresses DU145 cell growth, migration, and invasion.

<p>(A) <i>Hsa-miR-765</i> mimic effectively recognizes reporter with complementary sequence of <i>hsa-miR-765</i> in DU145 cells. Fold changes of luciferase activities of the <i>hsa-miR-765</i> mimic treated cells relative to the cells treated with the negative-control mimic are presented (n = 3). Transfection reagents were used as control. (B) <i>Hsa-miR-765</i> mimic reduces DU145 cell growth. MTS assay was performed on the cells treated with <i>hsa-miR-765</i> mimic or negative-control mimic or transfection control for 4 days (n = 8). (C) <i>Hsa-miR-765</i> mimic significant reduces G0/G1 to G2/M ratio in DU145. Representative DNA histograms (n = 3) are presented. (D) <i>Hsa-miR-765</i> mimic treatment causes up-regulation of cyclin A, cyclin B, and phosphorylated-cdc2 expression in DU145 cells. Protein expression levels of cell cycle regulator proteins were determined by Western blot analyses. Two independent experiments were performed and one representative set of data was presented. (E) <i>Hsa-miR-765</i> mimic suppresses DU145 cell migration and invasion as shown in transwell migration assay (top left) and invasion assay (top right), respectively. Representative micrographs of the cells after transwell migration (top left) or invasion assay (top right) are presented. Fold changes of migration (bottom left) and invasion (bottom right) of DU145 cells with either <i>hsa-miR-765</i> mimic or negative-control mimic relative to the control cells with negative-control mimic are presented (n = 3). (F) <i>Hsa-miR-765</i> mimic significantly reduces stress fibers and filopodia formations in DU145 cells. Representative micrographs and the percentages of the cells with intense stress fibers and the filopodial cells (n = 3) are presented. Student's t-test was used for comparisons with a cutoff p value of 0.05. ** p<0.01; bar = S.D.</p

HMGA1 is a direct target of <i>hsa-miR-765</i>.

<p>(A) The 3′UTR of <i>HMGA1</i> from +8910 to +8929 is predicted to be <i>hsa-miR-765</i> binding site. (B) <i>Hsa-miR-765</i> interacts with 3′UTR of <i>HMGA1</i> in a targeting reporter assay. DU145 cells were transfected with either pMIR-empty or pMIR-HMGA1-3UTR in which 3′ UTR of <i>HMGA1</i> (+8026–+9332) was cloned into the 3′ end of luciferase. Reporter activities of the pMIR-HMGA1-3UTR transfected cells treated with <i>hsa-miR-765</i> mimic or negative-control mimic are compared (n = 3). (C) <i>Hsa-miR-765</i> mimic reduced HMGA1 protein expression in DU145 cells. Protein and mRNA levels of HMGA1 in the <i>hsa-miR-765</i> mimic- and negative-control mimic-treated cells were determined by Western blot analysis (upper) and real-time RT-PCR analysis (lower), respectively. Results from <i>miR-765</i> mimic vs negative control mimic are compared (n = 3). (D) Fulvestrant reduces HMGA1 protein expression in DU145 cells. Protein level of HMGA1 and β-actin in the fulvestrant-treated and ethanol-treated control (CTL) cells were determined by Western blot analysis. (E) Ectopic expression of HMGA1 blocks fulvestrant-induced DU145 cell growth inhibition. The relative cell growth was determined after 4 days of treatment with fulvestrant or ethanol after stable transfection of <i>HMGA1</i> (or empty vector for control) for a week. Protein levels of HMGA1 were shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098037#pone.0098037.s006" target="_blank">Figure S6</a>. The cell growth of fulvestrant-treated cells with HMGA1 overexpression vs empty vector are compared (n = 8). Student's t-test was performed to determine significance between groups using a cutoff p value of 0.05. **p<0.01; bar = S.D.</p

Fulvestrant inhibits DU145 cell growth, migration, and invasion.

<p>(A) Fulvestrant induces growth inhibition of DU145 cells via an ERβ-dependent mechanism. Growth of the fulvestrant-treated DU145 cells with or without ERβ siRNA knockdown for 4 days relative to the ethanol-treated control cells with negative-control siRNA are presented and compared (n = 8). ERβ expression was also knocked down by another siRNA (siRNA#2) and the similar results were obtained (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098037#pone.0098037.s005" target="_blank">Figure S5</a>). (B) Fulvestrant induces DU145 cell-cycle arrest at G2/M phase. Representative DNA histograms of 48 hrs fulvestrant -or ethanol- (control) treated cells and percentage distributions of the cells at G0/G1 and G2/M phases (n = 3) are presented and compared. (C) Fulvestrant induces expression of G2/M markers. DU145 cells were treated with fulvestrant or ethanol for 2 days (control) and cell cycle markers were determined by Western blot analysis. Two independent experiments were performed and one representative set of data was presented. (D) Fulvestrant suppresses cell migration. A wound-healing assay was performed on the fulvestrant- and ethanol (EtOH)-treated DU145 cells (n = 3). Representative micrographs of the fulvestrant- and ethanol-treated cell cultures with scratches at 0 h and after 16 h are shown. The wound is marked by dotted lines. (E) Fulvestrant inhibits transwell migration (left panel) and invasion (right panel) in DU145 cells (n = 3) after 5 hrs of fulvestrant treatment. (F) Reductions of filopodial cells and cells with intense stress fibers by fulvestrant (treated with 48 hrs) via an ERβ-dependent mechanism. Representative micrographs and the percentages of the cells with intense stress fibers and the filopodial cells (n = 3) are presented. Student t-test was performed to determine significance with a cutoff p value of 0.05. ** p<0.01; bars = S.D.</p

ERβ is involved in fulvestrant-induced upregulation of <i>hsa-miR-765</i> expression.

<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

Significant reduction of HMGA1 protein correlates with enhanced expression of <i>hsa-miR-765</i> in fulvestrant-treated clinical PCa specimens.

<p>(A) Higher level of <i>hsa-miR-765</i> is detected in fulvestrant-treated clinical PCa specimens. Relative fold changes between expression of <i>hsa-miR-765</i> in the fulvestrant-treated (n = 7) and untreated (n = 7) clinical specimen are presented. Student's t-test was performed to determine significance between two groups. *p<0.05; bar = S.E.M. (B) Nuclear expression of HMGA1 and AR is reduced in fulvestrant-treated clinical PCa specimens. HMGA1 immunostaining was performed in the clinical PCa specimens from the fulvestrant-treated (n = 5) and untreated (n = 5) patients. Representative micrographs (100×) are shown. In upper panel, a magnified view (400×) of a selected region (dashed rectangle) in each micrograph is shown as a small insert to show the immunostaining of HGMA1 in the nuclei of Gleason grade 3/4 cancer foci. Imunnopositivity of nuclear AR is reduced in fulvestrant-treated Gleason grade 3/4 foci as shown in lower panel (400×). (C) Expression of both HMGA1 and AR is significantly reduced in fulvestrant-treated clinical PCa specimens when compared with their respective untreated samples (*p<0.05; **p<0.01; n = 9 (from 5 patients) for untreated samples; n = 10 (from 5 patients) for fulvestrant-treated samples, bar = S.E.M).</p