Generation of CRMP1 stably expressed MB cells.

<p>DAOY, ONS-76, and UW228-1 cells were transfected with pcDNA3.1-CRMP1 or pcDNA3.1 control plasmids. Stable clones were isolated by selection with G418. (A) Quantitative RT-PCR of CRMP1 in CRMP1 stably expressed cells and control cells. CRMP1 expression was normalized with GAPDH. The columns are expressed as mean±SD (** indicates p<0.01). (B) Western blot analysis of CRMP1 in clones of DAOY (top), ONS-76 (middle), and UW228-1 (bottom). The numbers under the protein bands indicated the relative band intensity normalized with ACTIN, and the control clones were set to 1. ImageJ software was used for quantification.</p

Effects of CRMP1 expression on invasion.

<p>Clones of (A) DAOY, (B) ONS-76, and (C) UW228-1 cells evaluated for invasion by matrigel invastion assay. Five random fields on each membrane were counted for the number of invaded cells. The bars illustrate mean±SD of the number of invaded cells in three separate experiments. On top of bar graphs shows representative photomicrographs. * indicates p<0.05; ** indicates p<0.01.</p

Expression of CRMP1 induced suppression of cell proliferation in MB cells.

<p>Clones of (A) DAOY, (B) ONS-76, and (C) UW228-1 were assessed for cell proliferation by MTT assay every 24h for a total of four days. Quadruplicates were performed in each experiment. The data represent the result of three individual experiments. * indicates p<0.05; ** indicates p<0.01.</p

An inverse correlation between HMGA1 and CRMP1 expression as revealed by expression profiling studies.

<p>Expression data were retrieval from (A) Cho et al. study which comprised of 189 MB and (B) Northcott et al. study which comprised of 103 MB. Correlation coefficients were determined with Pearson's correlation analysis.</p

Reduced transcript level of CRMP1 in MB tumors and cell lines.

<p>Expression of CRMP1 was measured by quantitative RT-PCR in 32 primary MBs (black bar), 8 cell lines (grey bar) and 5 normal cerebella (white bar). Compared to the mean of 5 normal cerebella, CRMP1 was significantly lower in 12/32 (37.5%) of MB and 8/8 (100%) of MB cell lines. The samples below the dot line were detected with reduced CRMP1 expression. The bar represents the mean of three measurements and the error bars indicates the standard deviation (SD).</p

Expression of CRMP1 inhibited cell migration.

<p>Clones of (A) DAOY, (B) ONS-76, and (C) UW228-1 stably expressing CRMP1 were examined for migration by transwell assay. The bars shown as mean±SD of the number of cells migrated across uncoated transwell membranes in three independent experiments. Representative images are shown on top of bar graphs. * indicates p<0.05; ** indicates p<0.01.</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

Cell-Free Urinary MicroRNA-99a and MicroRNA-125b Are Diagnostic Markers for the Non-Invasive Screening of Bladder Cancer

<div><p>Background</p><p>Evidence implicated the diagnostic significance of microRNAs in whole urine/urine sediments in urothelial carcinoma of the bladder (UCB). However, the contaminated blood cells in patients with haematouria significantly altered the expression profiles of urinary microRNA, influencing the test accuracy.</p><p>Methods</p><p>MicroRNA profiles of the urine supernatants of UCB patients and controls without any malignancy and profiles of malignant and corresponding normal mucosa tissues from the patients were determined by microRNA microarray and compared to identify differentially expressed microRNAs. The differential expression was verified in the tissues of an independent patient cohort by RT-qPCR. The diagnostic significance of selected microRNAs as biomarkers in the urine supernatant was investigated in the expanded cohorts.</p><p>Results</p><p>MicroRNA-99a and microRNA-125b were down-regulated in the urine supernatants of UCB patients. The degree of down-regulation was associated with the tumor grade. A diagnostic model was developed using a combined index of the levels of microRNA-99a and microRNA-125b in the urine supernatant with a sensitivity of 86.7%, a specificity of 81.1% and a positive predicted value (PPV) of 91.8%. Discriminating between high- and low-grade UCB, the model using the level of microRNA-125b alone exhibited a sensitivity of 81.4%, a specificity of 87.0% and a PPV of 93.4%.</p><p>Conclusions</p><p>The results revealed a unique microRNA expression signature in the urine supernatants of UCB patients for the development of molecular diagnostic tests. An effective cell-free urinary microRNA-based model was developed using a combined index of the levels of microRNA-99a and microRNA-125b to detect UCB with good discriminating power, high sensitivity and high specificity.</p></div

<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

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