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Background: Analysis of a microRNA (miRNA) expression signature of bladder cancer (BC) by deep-sequencing revealed that clustered miRNAs microRNA (miR)-451a, miR-144-3p, and miR-144-5p were significantly downregulated in BC tissues. We hypothesised that these miRNAs function as tumour suppressors in BC. The aim of this study was to investigate the functional roles of these miRNAs and their modulation of cancer networks in BC cells. Methods: The functional studies of BC cells were performed using transfection of mature miRNAs. Genome-wide gene expression analysis, in silico analysis, and dual-luciferase reporter assays were applied to identify miRNA targets. The association between miR-144-5p levels and expression of the target genes was determined, and overall patient survival as a function of target gene expression was estimated by the Kaplan–Meier method. Results: Gain-of-function studies showed that miR-144-5p significantly inhibited cell proliferation by BC cells. Four cell cycle-related genes (CCNE1, CCNE2, CDC25A, and PKMYT1) were identified as direct targets of miR-144-5p. The patients with high CCNE1 or CCNE2 expression had lower overall survival probabilities than those with low expression (P=0.025 and P=0.032). Conclusion: miR-144-5p functions as tumour suppressor in BC cells. CCNE1 and CCNE2 were directly regulated by miR-144-5p and might be good prognostic markers for survival of BC patients.

Recent clinical trials of chemotherapeutics for advanced bladder cancer (BC) have shown limited benefits. Therefore, new prognostic markers and more effective treatment strategies are required. One approach to achieve these goals is through the analysis of RNA networks. Our recent studies of microRNA (miRNA) expression signatures revealed that the microRNA-23b/27b (miR-23b/27b) cluster is frequently downregulated in various types of human cancers. However, the functional role of the miR-23b/27b cluster in BC cells is still unknown. Thus, the aim of the present study was to investigate the functional significance of the miR-23b/27b cluster and its regulated molecular targets, with an emphasis on its contributions to BC oncogenesis and metastasis. The expression levels of the miR-23b/27b cluster were significantly reduced in BC clinical specimens. Restoration of mature miR-23b or miR-27b miRNAs significantly inhibited cancer cell migration and invasion, suggesting that these clustered miRNAs function as tumor suppressors. Gene expression data and in silico analysis demonstrated that the genes coding for the epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (c-Met) were potential targets of the miR-23b/27b cluster. Luciferase reporter assays and western blotting demonstrated that EGFR and c-Met receptor trypsine kinases were directly regulated by these clustered miRNAs. We conclude that the decreased expression of the tumor-suppressive miR-23b/27b cluster enhanced cancer cell proliferation, migration and invasion in BC through direct regulation of EGFR and c-Met signaling pathways. Our data on RNA networks regulated by tumor-suppressive miR-23b/27b provide new insights into the potential mechanisms of BC oncogenesis and metastasis.

// Ryosuke Matsushita 1 , Hirofumi Yoshino 1 , Hideki Enokida 1 , Yusuke Goto 2 , Kazutaka Miyamoto 1 , Masaya Yonemori 1 , Satoru Inoguchi 1 , Masayuki Nakagawa 1 , Naohiko Seki 2 1 Department of Urology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan 2 Department of Functional Genomics, Chiba University Graduate School of Medicine, Chuo-ku, Chiba, Japan Correspondence to: Naohiko Seki, e-mail: naoseki@faculty.chiba-u.jp Keywords: miR-145-5p, miR-145-3p, tumor-suppressor, UHRF1, bladder cancer Received: February 22, 2016 Accepted: March 28, 2016 Published: April 09, 2016 ABSTRACT In microRNA (miRNA) biogenesis, the guide-strand of miRNA integrates into the RNA induced silencing complex (RISC), whereas the passenger-strand is inactivated through degradation. Analysis of our miRNA expression signature of bladder cancer (BC) by deep-sequencing revealed that microRNA ( miR ) -145-5p (guide-strand) and miR-145-3p (passenger-strand) were significantly downregulated in BC tissues. It is well known that miR-145-5p functions as a tumor suppressor in several types of cancer. However, the impact of miR-145-3p on cancer cells is still ambiguous. The aim of the present study was to investigate the functional significance of miR-145-3p and BC oncogenic pathways and targets regulated by miR-145-5p/miR-145-3p . Ectopic expression of either miR-145-5p or miR-145-3p in BC cells significantly suppressed cancer cell growth, migration and invasion and it also induced apoptosis. The gene encoding ubiquitin-like with PHD and ring finger domains 1 ( UHRF1 ) was a direct target of these miRNAs. Silencing of UHRF1 induced apoptosis and inhibited cancer cell proliferation, migration, and invasion in BC cells. In addition, overexpressed UHRF1 was confirmed in BC clinical specimens, and the high UHRF1 expression group showed a significantly poorer cause specific survival rate in comparison with the low expression group. Taken together, our present data demonstrated that both strands of miR-145 ( miR-145-5p : guide-strand and miR-145-3p : passenger-strand) play pivotal roles in BC cells by regulating UHRF1 . The identification of the molecular target of a tumor suppressive miRNAs provides novel insights into the potential mechanisms of BC oncogenesis and suggests novel therapeutic strategies.

INTRODUCTION AND OBJECTIVES: Genomic characterization of urothelial carcinoma of the bladder (UCB) has begun to reveal significant intertumor heterogeneity when comparing samples from different subjects. As in other malignancies, intratumor heterogeneity, which may allow for tumor evolution and adaption, poses a significant challenge to personalized-medicine strategies. METHODS: To examine UCB tumor evolution and heterogeneity, we performed next-generation targeted sequencing on multiple temporally and spatially separated bladder tumors obtained at time of transurethral resection (TUR) and radical cystectomy (RC). Specimens were analyzed using a next-generation, targeted sequencing assay designed to identify point mutations, indels, and copy number alterations in 300 cancer-associated genes. RESULTS: Phylogenetic reconstruction revealed evidence of branched evolutionary growth. Evaluation of multiple tumors from individual subjects identified both shared and unique potential driver mutations. Evidence of convergent phenotypic evolution was detected through analysis of multiple distinct tumors from several subjects. For example, three separate tumors in one subject (see Figure) shared a common PIK3CA mutation (E453K) and had unique second mutations in PIK3CA (E542V, E545K, and E545Q, respectively). In another subject, distinct inactivatingmutations of EP300were identified in two temporally separated tumor samples. Macrodissection of single tumors into non-invasive and invasive components revealed significant intratumor heterogeneity; one case illustrates howanalysis of amuscleinvasive TURspecimen could result in undersampling and therebymiss the tumor clone that persisted at time of RC. CONCLUSIONS: We demonstrate branched evolution of UCB through genomic analyses of multiple temporally and spatially distinct bladder tumors from individual subjects. Macrodissection of individual tumor samples identified significant intratumor heterogeneity. These concepts may present major challenges to personalized-medicine approaches that rely on sampling of a single tumor at a specific timepoint in the evolution of a patient’s UCB.

Here, we found that microRNA-24-1 (miR-24-1) is significantly reduced in bladder cancer (BC) tissues, suggesting that it functions as a tumour suppressor. Restoration of mature miR-24-1 inhibits cancer cell proliferation and induces apoptosis. Forkhead box protein M1 (FOXM1) is a direct target gene of miR-24-1, as shown by genome-wide gene expression analysis and luciferase reporter assay. Overexpressed FOXM1 is confirmed in BC clinical specimens, and silencing of FOXM1 induces apoptosis in cancer cell lines. Our data demonstrate that the miR-24-1–FOXM1 axis contributes to cancer cell proliferation in BC, and elucidation of downstream signalling will provide new insights into the molecular mechanisms of BC oncogenesis.