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significantly enhanced DPYSL3 gene expression in the mRNA and protein levels at day 3 after transfection in 22RV1, DU145 and PC-3 cells. CONCLUSIONS: Our data reveal that isoform-1 of tumor suppressor gene DPYSL3 expression is significantly reduced in malignant tissues and is responsive to up-regulation by a saRNA-based approach. Further animal based in vivo testing of the saRNAs is being undertaking by our group to evaluate their clinical significance as a novel gene therapy strategy.

In 2005, the International Society of Urological Pathology (ISUP) introduced several modifications to the original Gleason system that were intended to enhance the prognostic power of Gleason score (GS). The objective of this study was to clinically validate the 2005 ISUP Gleason grading system for its ability to detect metastasis. We queried our institutional RP database for men with NCCN clinically localized intermediate to high-risk disease undergoing radical prostatectomy (RP) between 1992 and 2010 with no additional treatment until the time of metastatic progression. A case-cohort design was utilized. A total of 333 available RP samples were re-reviewed and GS was reassigned per the 2005 ISUP Gleason system. Cumulative incidence of metastasis was 0%, 8.4%, 24.5% and 44.4% among specimens that were downgraded, unchanged, had one point GS increase and two point GS increase, respectively. The hazard ratio for metastasis raised in GS 8 and 9 compared to GS 7 from 2.77 and 5.91 to 3.49 and 9.31, respectively. The survival c-index of GS increased from 0.70 to 0.80 when samples were re-graded at 5 years post RP. The c-index of the reassigned GS was higher than the original GS (0.77 vs 0.64) for predicting PCSM at 10 years post RP. The regraded GS improved the prediction of metastasis and PCSM. This validates the updated Gleason grading system using an unambiguous clinical endpoint and highlights the need for reassignment of Gleason grading according to 2005 ISUP system when considering comparisons of novel biomarkers to clinicopathological variables in archival cohorts. PLOS ONE | DOI:10.1371/journal.pone.0146189 January 5, 2016 1 / 13 OPEN ACCESS Citation: Faraj SF, Bezerra SM, Yousefi K, Fedor H, Glavaris S, Han M, et al. (2016) Clinical Validation of the 2005 ISUP Gleason Grading System in a Cohort of Intermediate and High Risk Men Undergoing Radical Prostatectomy. PLoS ONE 11(1): e0146189. doi:10.1371/journal.pone.0146189 Editor: Xin Gao, Sun Yat-sen University, CHINA Received: October 18, 2015 Accepted: December 14, 2015 Published: January 5, 2016 Copyright: © 2016 Faraj et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: Kasra Yousefi and Elai Davicione are employees of GenomeDx Biosciences Inc. The remaining authors have no conflict of interest to declare. The funder provided support in the form of salaries for authors [KY and ED], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.

are still unclear. Our miRNA expression signature of prostate cancer (PCa) showed that miR-23b/27b/24-1 clustered miRNAs were significantly downregulated in PCa. The aim of the present study was to investigate the functional significance of the miR-23b/27b/24-1 cluster and to identify the molecular targets regulated by this cluster in PCa cells. METHODS: Gain-of-function studies using mature miRNAs were performed to investigate cell proliferation, migration and invasion in PCa cell lines. To identify the molecular targets potentially regulated by the miR-23b/27b/24-1 cluster, we applied gene expression data and in silico analysis using TargetScan database [http://www.targetscan. org/] and GENECODIS software. Loss-of-function assays were performed to investigate the functional significance of miR-23b/27b/24-1 cluster target genes. RESULTS: Restoration of miR-23b, miR-27b and miR-24-1 in PCa cell lines revealed that these miRNAs significantly inhibited cancer cell migration and invasion. Gene expression data and in silico analysis demonstrated that golgi membrane protein 1 (GOLM1) was regulated by these clustered miRNAs. Silencing of the target gene resulted in significant inhibition of cell proliferation, migration and invasion in PCa cells. CONCLUSIONS: The miR-23b/27b/24-1 clusterd miRNAs were frequently down-regulated and function as tumor suppressors in PCa. Elucidation of the molecular targets regulated by the tumorsuppressive clustered miRNAs will provide new insights into the potential molecular mechanisms of PCa oncogenesis and metastasis and will facilitate the development of novel therapeutic strategies for the treatment of PCa.