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Background: We hypothesized that the tissue components of in-stent restenosis (ISR) might differ between drug-eluting stents (DES) and bare-metal stents (BMS) and that these differences could be distinguished by qualitative and quantitative optical coherence tomography (OCT) analyses. Methods and Results: One-hundred and twenty-two initial ISR lesions (sirolimus-eluting stents: n=28; paclitaxel-eluting stents: n=51; BMS: n=43) were evaluated with OCT. Based on their OCT appearance, the lesions were classified as homogeneous, layered or heterogeneous. The optical properties of backscatter, attenuation and signal intensity of the neointimal tissue (NIT) were quantified. To evaluate the vascular response after balloon angioplasty (BA), the rate of reduction of the NIT area (NITA) was calculated (NITA before – after BA/NITA before BA at the minimum lumen cross-sectional area). Among the morphologic OCT patterns, the layered type was predominant with DES, whereas lesions were homogeneous with BMS (P

To propose an appropriate prophylactic antimicrobial therapy for patients undergoing brachytherapy, we evaluated the relationships between various antimicrobial prophylaxis (AMP) protocols and the incidence of postimplant infections in a multicenter cohort study conducted in Japan. The records of 826 patients with localized prostate cancer who underwent a transperineal (125)I brachytherapy procedure between January 2009 and December 2010 were retrospectively reviewed. Perioperative infections, including surgical site and remote infections, were recorded up to postoperative day 30. A total of 6 (0.73%) patients had a perioperative infection following seed implantation, of whom all received AMP for 1 or more days. None of the patients who received a single-dose protocol of AMP using fluoroquinolone p.o. or penicillin with a beta-lactamase inhibitor i.v. developed a perioperative infection. Statistical analysis showed that a single-dose protocol was more significantly related to a lower risk of perioperative infection as compared to the other AMP protocols examined (p = 0.045). Furthermore, our results indicated that bacteriuria and preoperative hair removal were risk factors of perioperative infection with statistical significance (p = 0.007, p = 0.004). Analysis of patient clinical parameters, including age, American Society of Anesthesiologists score, diabetes mellitus, prostate volume, numbers of implanted seeds and needle punctures, operation time, and indwelling duration time of the Foley catheter, did not reveal significant differences in terms of perioperative infection. Our results indicated that a single-dose AMP protocol is sufficient to prevent perioperative infections following seed implantation. On the other hand, AMP is only one of several measures to prevent perioperative infectious complications. It is necessary to know that the patient must have no bacteriuria and that preoperative hair removal should be avoided.

We previously demonstrated that miR-133a is a tumor-suppressive microRNA (miRNA) and is commonly down-regulated in human bladder cancer (BC). The aim of this study is to determine a novel oncogenic gene targeted by miR-133a in BC.To identify genes targeted by miR-133a, an oligo-microarray analysis was performed using the miR-133a-transfected BC cell lines. For gain/loss-of-function studies, miR-133a/si-glutathione S-transferase π1 (GSTP1)-transfectants were subjected to XTT assay and flow cytometry to evaluate their cell viability and apoptosis status. The luciferase reporter assay was used to confirm the actual binding sites between miR-133a and GSTP1 mRNA. The mRNA and protein expression of GSTP1 in BC cell lines and clinical samples were evaluated by real-time RT-PCR and Western blot, respectively.MiR-133a transfection induced cell viability inhibition and apoptosis in BC cell lines. We focused on the GSTP1 gene that was the top 7 down-regulated one in the gene profile from the miR-133a-transfectants. MiR-133a transfection repressed expression levels of mRNA and protein levels of GSTP1. A luciferase reporter assay suggested that the actual binding may occur between miR-133a and GSTP1 mRNA. Cell viability inhibition and apoptosis were induced in the si-GSTP1 transfectants compared with the controls (P0.005). GSTP1 mRNA expression levels in 43 clinical BCs were significantly higher than those in eight normal bladder epitheliums (P = 0.0277).Our data suggest that tumor suppressive miR-133a directly regulated oncogenic GSTP1 gene in BC, and that an anti-apoptotic effect mediated by GSTP1 is maintained by miR-133a down-regulation in human BC.

Aims We performed this study to clarify natural consequences of abnormal structures (stent malapposition, thrombus, tissue prolapse, and stent edge dissection) after percutaneous coronary intervention (PCI). Methods and results Thirty-five patients treated with 40 drug-eluting stents underwent serial optical coherence tomography (OCT) imaging immediately after PCI and at the 8-month follow-up. Among a total of 73 929 struts in every frame, 431 struts (26 stents) showed malapposition immediately after PCI. Among these, 49 remained malapposed at the follow-up examination. The mean distance between the strut and vessel wall (S–V distance) of persistent malapposed struts on post-stenting OCT images was significantly longer than that of resolved malapposed struts (342 ± 99 vs. 210 ± 49 μm; P

Recently, many studies have suggested that microRNAs (miRNAs) are involved in cancer cell development, invasion, and metastasis of various types of human cancers. In a previous study, miRNA expression signatures from renal cell carcinoma (RCC) revealed that expression of microRNA‐135a (miR‐135a) was significantly reduced in cancerous tissues. The aim of this study was to investigate the functional significance of miR‐135a and to identify miR‐135a‐mediated molecular pathways in RCC cells. Restoration of mature miR‐135a significantly inhibited cancer cell proliferation and induced G(0)/G(1) arrest in the RCC cell lines caki2 and A498, suggesting that miR‐135a functioned as a potential tumor suppressor. We then examined miR‐135a‐mediated molecular pathways using genome‐wide gene expression analysis and in silico analysis. A total of 570 downregulated genes were identified in miR‐135a transfected RCC cell lines. To investigate the biological significance of potential miR‐135a‐mediated pathways, we classified putative miR‐135a‐regulated genes according to the Kyoto Encyclopedia of Genes and Genomics pathway database. From our in silico analysis, 25 pathways, including the cell cycle, pathways in cancer, DNA replication, and focal adhesion, were significantly regulated by miR‐135a in RCC cells. Moreover, based on the results of this analysis, we investigated whether miR‐135a targeted the c‐MYC gene in RCC. Gain‐of‐function and luciferase reporter assays showed that c‐MYC was directly regulated by miR‐135a in RCC cells. Furthermore, c‐MYC expression was significantly upregulated in RCC clinical specimens. Our data suggest that elucidation of tumor‐suppressive miR‐135a‐mediated molecular pathways could reveal potential therapeutic targets in RCC.

Background: Previous studies have revealed that miR-26a-5p and miR-26b-5p act as tumour suppressors in various types of cancer tissues. Here, we aimed to investigate the functional roles of these miRNAs and to identify their regulatory targets in bladder cancer (BC). Methods: We performed functional assays in BC cells using transfection of mature microRNAs (miRNAs). In silico and luciferase reporter analyses were applied to identify target genes of these miRNAs. The overall survival (OS) of patients with BC was evaluated by the Kaplan–Meier method. Results: miR-26a-5p and miR-26b-5p were significantly downregulated in BC tissues. Restoration of these miRNAs inhibited cell migration and invasion in BC. The gene encoding procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2), a collagen crosslinking enzyme, was directly regulated by miR-26a-5p and miR-26b-5p. Kaplan–Meier analysis revealed that patients with high PLOD2 expression had significantly shorter OS compared with those with low PLOD2 expression (P=0.0153). Conclusions: PLOD2, which is associated with the stiffness of the extracellular matrix, was directly regulated by miR-26a-5p and miR-26b-5p and may be a good prognostic marker in patients with BC.

// 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.

The ultrasonic horns with cylindrical concave surfaces (concaved horn) have been designed and the effect of the concave on cavitation bubbles generation in water has been investigated. High speed camera observation was made on the vibrating horn tip in water and the generation behavior and the impact force of the cavitation bubbles were investigated. In the measurement of impact force, load vibration occurred as the cavitation bubbles collapsed, and peak frequencies were observed at the vibratory frequency of the horn and its harmonic frequency. The impact force was evaluated based on the above peak frequency. The result showed that the concaved horn provide the higher impact force compared to the horn without the concave. Frequency analysis of the load vibration showed that the concaved horn enhanced the generation of cavitation bubbles in the number. The distribution of cavitation bubbles on the horn-tip was evaluated using high-speed image. It was confirmed that the cavitation bubbles flow was effectively converged to the center of concave.

There is an expectation that in the near future there will be an effective change in the Brazilian accounting professional behavior. The accounting language is of legal, economic and social nature and is expected to contribute for better decisions in terms of economic value creation and financial management in an organization. The theory of communication is important for accountants in Brazil. If the accounting is to be considered as a business language the accountants must to have knowledge on the theory of semiotics and communication.

microRNAs (miRNAs) have key roles in human tumorigenesis, tumor progression and metastasis. miRNAs are aberrantly expressed in many human cancers and can function as tumor suppressors or oncogenes that target many cancer-related genes. This study seeks to identify novel miRNA-regulated molecular pathways in prostate cancer (PCa). The miRNA expression signature in clinical specimens of PCa showed that 56 miRNAs were significantly downregulated in PCa compared with non-PCa tissues. We focused on the top four downregulated miRNAs (miR-187, miR-205, miR-222 and miR-31) to investigate their functional significance in PCa cells. Expression levels of these four miRNAs were validated in PCa specimens (15 PCa tissues and 17 non-PCa tissues) to confirm that they were significantly reduced in these PCa tissues. Gain-of-function analysis demonstrated that miR-222 and miR-31 inhibited cell proliferation, invasion and migration in PCa cell lines (PC3 and DU145), suggesting that miR-222 and miR-31 may act as tumor suppressors in PCa. Genome-wide gene expression analysis using miR-222 or miR-31 transfectants to identify the pathways they affect showed that many cancer-related genes are regulated by these miRNAs in PC3 cells. Identification and categorization of the molecular pathways regulated by tumor suppressive miRNAs could provide new information about the molecular mechanisms of PCa tumorigenesis.

Many studies have recently suggested that microRNAs (miRNAs) contribute to the development of various types of human cancers as well as to their invasive and metastatic capacities. Previously, our miRNA expression signature of renal cell carcinoma (RCC) revealed that microRNA‑138 (miR‑138) was significantly reduced in cancer cells. The aim of the present study was to investigate the functional significance of miR‑138 and to identify its target genes in RCC cells. Restoration of mature miR‑138 in two RCC cell lines (A498 and 786‑O) caused changes in the bleb-like cell morphology, characteristics of the epithelial-mesenchymal transition (EMT). Restoration also significantly inhibited migration and invasion in the two RCC cell lines, suggesting that miR‑138 functions as a tumor suppressor. Genome-wide gene expression analysis (miR‑138 transfectants and RCC clinical specimens) and TargetScan database studies showed that vimentin (VIM) is a promising candidate target gene of miR‑138. It is well known that VIM is one of the most widely expressed mammalian intermediate filament proteins. Recent studies showed that VIM functions in cell adhesion, migration, survival and cell signaling processes via dynamic assembly/disassembly in cancer cells. We focused on VIM and investigated whether VIM was regulated by tumor suppressive miR‑138 and contributed to cancer cell migration and invasion in RCC cells. Restoration of miR‑138 in RCC cell lines suppressed VIM expression at both the mRNA and protein levels. Silencing studies of VIM in RCC cell lines demonstrated significant inhibition of cell migration and invasion activities in si-VIM transfectants. In clinical specimens of RCC, the expression levels of VIM were significantly upregulated in cancer tissues compared to adjacent non-cancerous tissues. Furthermore, immunohistochemistry showed that VIM expression levels in RCC specimens were significantly higher than those in normal renal tissues. These data suggest that VIM may function as an oncogene and is regulated by tumor suppressive miR‑138. The existence of a tumor suppressive miR‑138-mediated oncogenic pathway provides new insights into the potential mechanisms of RCC oncogenesis and metastasis.

Our previous study demonstrated that fascin homolog 1 (FSCN1) might have an oncogenic function in bladder cancer (BC) and that its expression was regulated by specific microRNAs (miRNAs). Recently, LIM and SH3 protein 1 (LASP1) as well as FSCN1 have been reported as actin filament bundling proteins in the same complexes attached to the inner surfaces of cell membranes. We hypothesize that LASP1 as well as FSCN1 have an oncogenic function and that is regulated by miRNAs targeting LASP1 mRNA.The expression levels of LASP1 mRNA in 86 clinical samples were evaluated by real-time RT-PCR. LASP1-knockdown BC cell lines were transfected by siRNA in order to examine cellular viability by XTT assay, wound healing assay, and matrigel invasion assay. We employed web-based software in order to search for candidate miRNAs targeting LASP1 mRNA, and we focused on miR-1, miR-133a, miR-145, and miR-218. The luciferase reporter assay was used to confirm the actual binding sites between the miRNAs and LASP1 mRNA.Real-time RT-PCR showed that LASP1 mRNA expression was higher in 76 clinical BC specimens than in 10 normal bladder epitheliums (P0.05). Loss-of-function studies using si-LASP1-transfected BC cell lines demonstrated significant cell viability inhibition (P0.0005), cell migration inhibition (P0.0001), and a decrease in the number of invading cells (P0.005) in the transfectants compared with the controls. Transient transfection of three miRNAs (miR-1, miR-133a, and miR-218), which were predicted as the miRNAs targeting LASP1 mRNA, repressed the expression levels of mRNA and protein levels of LASP1. The luciferase reporter assay demonstrated that the luminescence intensity was significantly decreased in miR-1, miR-133a, and miR-218 transfectants (P0.05), suggesting that these miRNAs have actual target sites in the 3' untranslated region of LASP1 mRNA. Furthermore, significant cell viability inhibitions occurred in miR-218, miR-1, and miR-133a transfectants (P0.001).Our data indicate that LASP1 may have an oncogenic function and that it might be regulated by miR-1, miR-133a, and miR-218, which may function as tumor suppressive miRNAs in BC.

Purpose The aim of this study was to find a novel molecular network involved in renal cell carcinoma (RCC) development through investigating the functions of miR-1 and miR-133a and their target genes. Methods We checked the expression levels of miR-1 and miR-133a in RCC cell lines and specimens (N = 40) using real time RT-PCR. MiR-1 and miR-133a transfectants were subjected to a gain-of-function study to identify the functions of the miRNAs. To find the target genes of the miRNAs, we analysed the gene expression profile of their transfectants and performed a luciferase reporter assay. mRNA expression levels of the candidate target gene in the clinical specimens were examined, and loss-of-function studies were performed. Results The expression levels of miR-1 and miR-133a were significantly suppressed in RCC cell lines and specimens. Ectopic restoration of miR-1 and miR-133a showed significant inhibition of cell proliferation and invasion, and moreover, revealed induction of apoptosis and cell cycle arrest. The luciferase assay revealed transgelin-2 (TAGLN2), selected as a target gene for miR-1 and miR-133a on the basis of the gene expression profile, to be directly regulated by both miR-1 and miR-133a. The loss-of-function studies showed significant inhibitions of cell proliferation and invasion in the si-TAGLN2 transfectant. The expression level of TAGLN2 mRNA was significantly up-regulated in the RCC specimens; in addition, there was a statistically significant inverse correlation between TAGLN2 and miR-1 and miR-133a expression. Conclusions Our data indicate that up-regulation of the oncogenic TAGLN2 was due to down-regulation of tumour-suppressive miR-1 and miR-133a in human RCC.

Hideo Hidaka 1,* , Naohiko Seki 2,* , Hirofumi Yoshino 1 , Takeshi Yamasaki 1 , Yasutoshi Yamada 1 , Nijiro Nohata 2 , Miki Fuse 2 , Masayuki Nakagawa 1 and Hideki Enokida 1 1 Department of Urology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan 2 Department of Functional Genomics, Graduate School of Medicine, Chiba University, Chiba, Japan * Denotes equal contribution Received: December 31, 2011; Accepted: January 21, 2012; Published: January 30, 2012; Keywords: microRNA, expression signature, miR-1285, tumor suppressor, renal cell carcinoma, transglutaminase 2 Correspondence: Hideki Enokida, email: // // Abstract MicroRNAs (miRNA) are non-coding RNAs, approximately 22 nucleotides in length, which function as post-transcriptional regulators. A large body of evidence indicates that miRNAs regulate the expression of cancer-related genes involved in proliferation, migration, invasion, and metastasis. The aim of this study was to identify novel cancer networks in renal cell carcinoma (RCC) based on miRNA expression signatures obtained from RCC clinical specimens. Expression signatures revealed that 103 miRNAs were significantly downregulated (< 0.5-fold change) in RCC specimens. Functional screening (cell proliferation assays) was performed to identify tumor suppressive activities of 20 downregulated miRNAs. Restoration of mature miRNAs in cancer cells showed that 14 miRNAs ( miR-1285 , miR-206 , miR-1 , miR-135a , miR-429 , miR-200c, miR-1291, miR-133b, miR-508-3p, miR-360-3p, miR-509-5p, miR-218, miR-335, miR-1255b and miR-1285 ) markedly inhibited cancer cell proliferation, suggesting that these miRNAs were candidate tumor suppressive miRNAs in RCC. We focused on miR-1285 because it significantly inhibited cancer cell proliferation, invasion, and migration following its transfection. We addressed miR-1285 -regulated cancer networks by using genome-wide gene expression analysis and bioinformatics. The data showed that transglutaminase 2 ( TGM2 ) was directly regulated by miR-1285 . Silencing of the target gene demonstrated significant inhibition of cell proliferation and invasion in the RCC cells. Furthermore, immunohistochemistry showed that TGM2 expression levels in RCC specimens were significantly higher than those in normal renal tissues. Downregulation of tumor suppressive miR-1285 , which targets oncogenic genes including TGM2, might contribute to RCC development. Thus, miR-1285 modulates a novel molecular target and provides new insights into potential mechanisms of RCC oncogenesis.

We have previously found that restoration of tumor suppressive microRNA-1 (miR-1), induced cell apoptosis in bladder cancer (BC) cell lines. However, the apoptosis mechanism induced by miR-1 was not fully elucidated. Alternative splicing of mRNA precursors provides cancer cells with opportunities to translate many oncogenic protein variants, which promote cell proliferation and survival under unpreferable condition for cancer development. Serine/arginine-rich (SR) protein family, which involved in alternative pre-mRNA splicing, plays a critical role for regulating apoptosis by splicing apoptosis-related genes. However, transcriptional regulation of SR proteins, themselves, has not been elucidated. In this study, we focused on splicing factor serine/arginine-rich 9 (SRSF9/SRp30c) on the basis of our previous genome-wide gene expression analysis using miR-1-transfected BC cell lines because putative target sites of miR-1 are existed in 3'-untranslated region (UTR) of SRSF9 mRNA. The expression levels of mRNA of SRSF9 were extremely reduced in the miR-1 transfectants. A luciferase activity significantly decreased in the transfectants suggesting that actual binding occurred between miR-1 and 3'UTR of SRSF9 mRNA. Loss-of-function assays demonstrated that significant inhibitions of cell proliferation, migration, and invasion were observed in the si-SRSF9 transfectants. Apoptosis assays demonstrated that cell apoptosis fraction increased and that caspase-3/7 was activated in the si-SRSF9 transfectants. Our data indicated that tumor suppressive miR-1 induces apoptosis through direct inhibition of SRSF9 in BC. The identification of molecular mechanisms between miRNAs and SR proteins could provide novel apoptosis pathways and their epigenetic regulations and offer new strategies for BC treatment.

Mobile thin-client services are gaining lots of attention from companies who concern about the security yet recognize the benefits of mobile computing in their businesses. The service is based on a private cloud system hosting virtual machines that can execute mobile OS instances. The owner of the private cloud needs to prepare sufficient server resources for hosting those virtual machines. In this paper, we propose a framework to guide server procurement decisions in a private cloud for mobile thin-client service, which aims to minimize the cost of unused servers while avoiding service level violation due to the lack of resources. To make a timely server procurement decision, the framework combines the techniques for workload estimation to individual VMs, demand estimation of newly created VMs and repetitive simulation of VM replacement algorithm. Through a simulation study, we show that the proposed framework can reduce the cost of unused servers by 25% while satisfying a service level, compared with a time-based heuristic decision method.

Objectives: Coincident with their worldwide use, resistance to fluoroquinolones in Escherichia coli has increased. To identify the gene expression profiles underlying fluoroquinolone resistance, we carried out genome-wide transcriptome analysis of fluoroquinolone-sensitive E. coli. Methods: Four fluoroquinolone-sensitive E. coli and five fluoroquinolone-resistant E. coli clinical isolates were subjected to complementary deoxyribonucleic acid microarray analysis. Some upregulated genes’ expression was verified by real-time polymerase chain reaction using 104 E. coli clinical isolates, and minimum inhibitory concentration tests were carried out by using their transformants. Results: A total of 40 genes were significantly upregulated in fluoroquinolone-resistant E. coli isolates (P

Background: Our recent analyses of miRNA expression signatures showed that miR-1 and miR-133a were significantly reduced in several types of cancer. Interestingly, miR-1 and miR-133a are located on the same chromosomal locus in the human genome. We examined the functional significance of miR-1 and miR-133a in prostate cancer (PCa) cells and identified the novel molecular targets regulated by both miR-1 and miR-133a. Methods and Results: The expression levels of miR-1 and miR-133a were significantly downregulated in PCa compared with non-PCa tissues. Restoration of miR-1 or miR-133a in PC3 and DU145 cells revealed significant inhibition of proliferation, migration, and invasion. Molecular target identification by genome-wide gene expression analysis and luciferase reporter assay showed that purine nucleoside phosphorylase (PNP) was directly regulated by both miRNAs. Silencing of the PNP gene inhibited proliferation, migration, and invasion in both PC3 and DU145 cells. Immunohistochemistry detected positive staining of PNP in PCa specimens. Conclusions: Downregulation of miR-1 and miR-133a was a frequent event in PCa and both function as tumour suppressors. The PNP is a novel target gene of both miRNAs and potentially functions as an oncogene. Therefore, identification of novel molecular networks regulated by miRNAs may provide new insights into the underlying causes of PCa oncogenesis.

Background: On the basis of the microRNA (miRNA) expression signature of maxillary sinus squamous cell carcinoma (MSSCC), we found that miR-874 was significantly reduced in cancer cells. We focused on the functional significance of miR-874 in cancer cells and identification of miR-874-regulated novel cancer networks in MSSCC. Methods: We used PCR-based methods to investigate the downregulated miRNAs in clinical specimens of MSSCC. Our signature analyses identified 23 miRNAs that were significantly reduced in cancer cells, such as miR-874, miR-133a, miR-375, miR-204, and miR-1. We focused on miR-874 as the most downregulated novel miRNA in our analysis. Results: We found potential tumour suppressive functions such as inhibition of cancer cell proliferation and invasion. A molecular target search of miR-874 revealed that PPP1CA was directly regulated by miR-874. Overexpression of PPP1CA was observed in MSSCC clinical specimens. Silencing of the PPP1CA gene significantly inhibited cancer cell proliferation and invasion. Conclusion: The downregulation of miR-874 was a frequent event in MSSCC, which suggests that miR-874 functions as a tumour suppressive miRNA, directly regulating PPP1CA that has a potential role of an oncogene. The identification of novel miR-874-regulated cancer pathways could provide new insights into potential molecular mechanisms of MSSCC oncogenesis.

Our microRNA (miRNA) expression signatures of hypopharyngeal squamous cell carcinoma, maxillary sinus squamous cell carcinoma and esophageal squamous cell carcinoma revealed that miR-375 was significantly reduced in cancer tissues compared with normal epithelium. In this study, we focused on the functional significance of miR-375 in cancer cells and identification of miR-375-regulated novel cancer networks in head and neck squamous cell carcinoma (HNSCC). Restoration of miR-375 showed significant inhibition of cell proliferation and induction of cell apoptosis in SAS and FaDu cell lines, suggesting that miR-375 functions as a tumor suppressor. We adopted genome-wide gene expression analysis to search for miR-375-regulated molecular targets. Gene expression data and luciferase reporter assays revealed that AEG-1/MTDH was directly regulated by miR-375. Cancer cell proliferation was significantly inhibited in HNSCC cells transfected with si-AEG-1/MTDH. In addition, expression levels of AEG-1/MTDH were significantly upregulated in cancer tissues. Therefore, AEG-1/MTDH may function as an oncogene in HNSCC. The identification of novel tumor suppressive miRNA and its regulated cancer pathways could provide new insights into potential molecular mechanisms of HNSCC oncogenesis.

BACKGROUND MiR-145 is down-regulated in various human cancers. We previously demonstrated that some actin-binding proteins were targeted by several microRNAs (miRNAs), including miR-145, in bladder and prostate cancer (CaP). The aim of this study is to determine a novel oncogenic gene targeted by miR-145 by focusing on actin-binding proteins in CaP. METHODS We focused on the SWAP switching B-cell complex 70 kDa subunit (SWAP70), which is an F-actin binding protein involved in activating B-cell transformation. A luciferase reporter assay was used to identify the actual binding sites between miR-145 and SWAP70 mRNA. Cell viability was evaluated by cell proliferation, wound healing, and matrigel invasion assays in si-SWAP70 transfectants. A total of 75 clinical prostate specimens were subjected to immunohistochemistry of SWAP70. RESULTS Molecular target searches of this miRNA and the luciferase reporter assay showed that SWAP70 was directly regulated by miR-145. Silencing of SWAP70 studies demonstrated significant inhibitions of cell migration and invasion in CaP cell lines. The SWAP70 positive-staining was significantly higher in percentage in the CaP than in benign prostate hyperplasia tissue. CONCLUSIONS Down-regulation of miR-145 was a frequent event in CaP, and it may have a tumor suppressive function. SWAP70 may be a target of miR-145, and it might have a potential oncogenic function. The novel molecular networks though which miR-145 acts, may provide new insights into the underlying molecular mechanisms of CaP. Prostate 71:1559–1567, 2011. © 2011 Wiley-Liss, Inc.

Based on the microRNA (miRNA) expression signatures of hypopharyngeal and esophageal squamous cell carcinoma, we found that miR-1 was significantly down-regulated in cancer cells. In this study, we investigated the functional significance of miR-1 in head and neck squamous cell carcinoma (HNSCC) cells and identified miR-1-regulated novel cancer pathways. Gain-of-function studies using miR-1 revealed significant decreases in HNSCC cell proliferation, invasion, and migration. In addition, the promotion of cell apoptosis and cell cycle arrest was demonstrated following miR-1e transfection of cancer cells. A search for the targets of miR-1 revealed that transgelin 2 (TAGLN2) was directly regulated by miR-1. Silencing of TAGLN2 significantly inhibited cell proliferation and invasion in HNSCC cells. Down-regulation of miR-1 and up-regulation of TAGLN2 were confirmed in HNSCC clinical specimens. Our data indicate that TAGLN2 may have an oncogenic function and may be regulated by miR-1, a tumor suppressive miRNA in HNSCC. The identification of novel miR-1-regulated cancer pathways could provide new insights into potential molecular mechanisms of HNSCC carcinogenesis.

A new diagnostic marker for urothelial carcinoma (UC) is needed to avoid painful cystoscopy during the initial diagnosis and follow-up period. However, the current urine markers are useless because of the low sensitivities and specificities for UC detection. MiR-96 and miR-183 were differentially upregulated microRNA in our previous microRNA screening for UC. The expression levels of miR-96 and miR-183 in the urine samples were significantly higher in 100 UC than in healthy controls (miR-96, P=0.0059; and miR-183, P=0.0044). The receiver-operating characteristic curve analyses demonstrated that each microRNA had good sensitivity and specificity for distinguishing UC patients from non-UC patients (miR-96, 71.0% and 89.2%; and miR-183, 74.0% and 77.3%). Our cohort included 78 UC patients who had undergone urinary cytology. MiR-96 was positively detected in 27 of 44 patients who had had a "negative" urinary cytology diagnosis. We combined the miR-96 detection data with the urinary cytology data, and diagnosed 61 of 78 cases as UC; sensitivity rose from 43.6% to 78.2%. We found significant stepwise increases in miR-96 and miR-183 expression with advancing tumor grade (miR-96, P=0.0057; and miR-183, P=0.0036) and pathological stage (miR-96, P=0.0332; and miR-183, P=0.0117). The expression levels of the microRNA were significantly lower in urine collected after surgery (miR-96, P=0.0241; and miR-183, P=0.0045). In conclusion, miR-96 and miR-183 in urine are promising tumor markers for UC. In particular, miR-96 may be a good diagnostic marker in combination with urinary cytology.

Secreted frizzled-related protein-5 (sFRP-5) has been identified as 1 of the secreted antagonists that bind Wnt protein. However, the functional significance of sFRP-5 in renal cell cancer (RCC) has not been reported. We hypothesized that sFRP-5 may be epigenetically downregulated through DNA methylation and histone modification and function as a tumor suppressor gene in RCC. Using tissue microarray and real-time RT-PCR, we found that sFRP-5 was significantly downregulated in kidney cancer tissues and cell lines, respectively. DNA bisulfite sequencing of the sFRP-5 promoter region in RCC cell lines showed it to be densely methylated, whereas there was few promoter methylation in normal kidney. The sFRP-5 expression was restored and the acetylation of H3 and H4 histones associated with the sFRP-5 promoter region were significantly increased after treatment with demethylation agent (5-Aza-dc) and histone deacetylase inhibitor (TSA). When RCC cells were transfected with the sFRP-5 gene, significant inhibition of anchorage independent colony formation and cell invasion were observed compared to controls. The sFRP-5 transfection also significantly induced apoptosis in RCC cells. In conclusion, this is the first report documenting that the sFRP-5 is downregulated by promoter methylation and histone acetylation and functions as a tumor suppressor gene by inducing apoptosis in RCC cells.

Background: The aim of this study is to find a novel molecular target based on chromosomal alteration and array-based gene expression analyses in bladder cancer (BC). We investigated a cancer testis antigen, LY6K, which is located on chromosome 8q24.3. Methods: Five BC cell lines were subjected to high-resolution array-comparative genomic hybridisation with 244 000 probes. The expression levels of LY6K mRNA were evaluated in BC cell lines and clinical BC specimens by real-time reverse transcription–PCR. The cell lines were subjected to fluorescence in situ hybridisation of LY6K. Cell viability was evaluated by cell growth, wound healing, and matrigel invasion assays. Results: Typical gained loci (P

214 Background: A novel cancer vaccine consisting of 20 mixed peptides (KRM-20) was designed to induce cytotoxic T lymphocytes (CTL) against twelve different tumor-associated antigens. The aim of this phase 2 trial was to examine whether KRM-20 in combination with docetaxel and dexamethasone enhances anti-tumor effects in patients with castration-resistant prostate cancer (CRPC). Methods: In this double-blind, placebo-controlled, randomized, phase 2 study, we enrolled chemotherapy-naïve patients with progressive CRPC from 10 medical centers in Japan. Eligible patients were randomly assigned 1:1 centrally to either KRM-20 combined with docetaxel and dexamethasone or placebo with docetaxel and dexamethasone. Patients initially received 5 weekly subcutaneous KRM-20 (20 mg) or placebo injections with daily oral dexamethasone (1 mg) following 5 courses of docetaxel (70 mg/m2 every 3 weeks). The primary endpoint was to compare each treatment for prostate-specific antigen (PSA) decline. Results: Between Jul 31, 2013 and Jul 11, 2014, 51 patients were enrolled to the trial: 25 were assigned to the KRM-20 arm, and 26 were allocated to the placebo arm. Mean % PSA levels from baseline in the KRM-20 arm significantly decreased during the treatment compared witht those in the placebo arm ( P = 0.028, MANOVA). Human leukocyte antigen matched peptide-specific immunoglobulin G ( P = 0.018) and CTL ( P = 0.007) responses in the KRM-20 arm significantly increased after the treatment. The appearance of myeloid-derived suppressor cells in the KRM-20 arm significantly decreased after the treatment ( P = 0.007). The addition of KRM-20 did not result in increased toxicity. There was no between-group differences in progression-free or overall survival. Conclusions: These findings suggest potential clinical benefits for the combined use of KRM-20 with docetaxel and dexamethasone in patients with CRPC. Clinical trial information: UMIN000011028.

185 Background: Patients with high-risk prostate cancer (PCa) according to D’Amico risk categories are prone to a pathological diagnosis of positive margins or lymph node invasion and biochemical recurrence, despite having undergone radical prostatectomy (RP). Therefore, it is controversial whether RP should be done for high risk PC patients. Methods: 87 high-risk PCa patients prospectively underwent ‘extended’ RP following neoadjuvant chemohormonal therapy (NAC); primarily 6 months of estramustine phosphate 280 mg bid, along with a LH-RH agonist/antagonist. Our surgical technique was developed to reduce the rates of positive surgical margins. The goal is to approach the muscle layer of the rectum by dissecting the mesorectal fascia and continuing the dissection through the mesorectum until the muscle layer of the rectum is exposed. The procedure was safely performed as a result of good recognition of the structure between the perineal body and the rectal surface. We also performed extended lymphadenectomy if the patients meet two or more of D’Amico risk categories Results: More than 1 year had elapsed after surgery in 69 of the 87 patients with the median follow-up period of 36.2 months. Among those 69 patients, 18 (26.1%) experienced PSA failure. Kaplan-Meier analyses revealed that significant poorer PSA progression-free survival were observed in patients with higher positive biopsy core ratio, lymph node metastasis, and higher pathological stage (pT3a/b). Multivariate Cox-regression analysis revealed that higher pathological stage (pT3a/b) was the only independent valuable for predicting PSA progression failure. These 18 cases received salvage androgen deprivation therapy followed-by external beam radiotherapy and showed no progression after the salvage therapies (median follow-up period, 34.6 months after PSA progression). Conclusions: NAC concordant with extended RP is feasible and contributes to negative surgical margins that might provide good cancer control for patients with high-risk PCa.

Background: We have recently identified down-regulated microRNAs including miR-145 and miR-133a in bladder cancer (BC). The aim of this study is to determine the genes targeted by miR-145, which is the most down-regulated microRNA in BC. Methods: We focused on fascin homologue 1 (FSCN1) from the gene expression profile in miR-145 transfectant. The luciferase assay was used to confirm the actual binding sites of FSCN1 mRNA. Cell viability was evaluated by cell growth, wound-healing, and matrigel invasion assays. BC specimens were subjected to immunohistochemistry of FSCN1 and in situ hybridisation of miR-145. Results: The miR-133a as well as miR-145 had the target sequence of FSCN1 mRNA by the database search, and both microRNAs repressed the mRNA and protein expression of FSCN1. The luciferase assay revealed that miR-145 and miR-133a were directly bound to FSCN1 mRNA. Cell viability was significantly inhibited in miR-145, miR-133a, and si-FSCN1 transfectants. In situ hybridisation revealed that miR-145 expression was markedly repressed in the tumour lesion in which FSCN1 was strongly stained. The immunohistochemical score of FSCN1 in invasive BC (n=46) was significantly higher than in non-invasive BC (n=20) (P=0.0055). Conclusion: Tumour suppressive miR-145 and miR-133a directly control oncogenic FSCN1 in BC.

MicroRNAs (miRNAs) are small noncoding RNAs that negatively regulate protein-coding genes. To identify miRNAs that have a tumor suppressive function in bladder cancer (BC), 156 miRNAs were screened in 14 BCs, 5 normal bladder epithelium (NBE) samples and 3 BC cell lines. We identified a subset of 7 miRNAs (miR-145, miR-30a-3p, miR-133a, miR-133b, miR-195, miR-125b and miR-199a*) that were significantly downregulated in BCs. To confirm these results, 104 BCs and 31 NBEs were subjected to real-time RT-PCR-based experiments, and the expression levels of each miRNA were significantly downregulated in BCs (p0.0001 in all). Receiver-operating characteristic curve analysis revealed that the expression levels of these miRNAs had good sensitivity (70%) and specificity (75%) to distinguish BC from NBE. Our target search algorithm and gene-expression profiling in BCs (Kawakami et al., Oncol Rep 2006;16:521-31) revealed that Keratin7 (KRT7) mRNA was a common target of the downregulated miRNAs, and the mRNA expression levels of KRT7 were significantly higher in BCs than in NBEs (p = 0.0004). Spearman rank correlation analysis revealed significant inverse correlations between KRT7 mRNA expression and each downregulated miRNA (p0.0001 in all). Gain-of-function analysis revealed that KRT7 mRNA was significantly reduced by transfection of 3 miRNAs (miR-30-3p, miR-133a and miR-199a*) in the BC cell line (KK47). In addition, significant decreases in cell growth were observed after transfection of 3 miRNAs and si-KRT7 in KK47, suggesting that miR-30-3p, miR-133a and miR-199a* may have a tumor suppressive function through the mechanism underlying transcriptional repression of KRT7.

We report an unusual case of bilateral, synchronous breast cancer in a male patient who had a history of estrogen therapy for prostate cancer. A 64-year-old Japanese man was diagnosed with T1N0M0 prostate cancer and received a total prostatectomy. Twenty months after the resection, the patient developed multiple bone metastases, and received radiation therapy, systemic chemotherapy, and hormone therapy for 15 months. After completing this treatment, he was diagnosed with T1N0M0 primary breast cancer in his left breast and underwent a modified mastectomy. Five months after the mastectomy he received systemic chemotherapy followed by estrogen therapy because of the progression of prostate cancer. Three months after this treatment, he was diagnosed with T1N0M0 primary breast cancer in his right breast. To the best of our knowledge, this is a rare case of synchronous bilateral male breast cancer following hormone therapy for prostate cancer.