Kagoshima-shi, Japan
Kagoshima-shi, Japan

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Matsuda R.,Kagoshima University | Enokida H.,Kagoshima University | Chiyomaru T.,Kagoshima University | Kikkawa N.,Chiba University | And 10 more authors.
British Journal of Cancer | Year: 2011

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 < 0.0001) at 6p21.33-p21.32, 8q24.3, 9q34.13, 11q13.1-q14.1, 12q13.12-q13.13, 16p13.3, and 20q11.21-q13.33 were observed in all of the cell lines. We focused on 8q24.3 locus where LY6K gene harbours, and it was the top upregulated one in the gene profile from the BC cell line. LY6K mRNA expression was significantly higher in 91 BCs than in 37 normal bladder epitheliums (P <0.0001). Fluorescence in situ hybridisation validated that the high LY6K mRNA expression was due to gene amplification in the region where the gene harbours. Cell viability assays demonstrated that significant inhibitions of cell growth, migration, and invasion occured in LY6K knock down BC cell lines; converse phenomena were observed in a stable LY6K transfectant; and LY6K knockdown of the transfectant retrieved the original phenotype from the LY6K transfectant. Conclusion: Upregulation of the oncogenic LY6K gene located on the gained locus at 8q24.3 may contribute BC development. © 2011 Cancer Research UK All rights reserved.


Toki K.,Kagoshima University | Enokida H.,Kagoshima University | Kawakami K.,Kagoshima University | Chiyomaru T.,Kagoshima University | And 7 more authors.
International Journal of Oncology | Year: 2010

We have previously reported a simple technique that combines microarray data from clinical bladder cancer (BC) specimens with those from a BC cell line (BOY) treated with a pharmacological demethylating agent [5-aza-2′- deoxycytidine (5-aza-dC)] to find candidate genes that have tumor suppressive functions. We focused on the cellular retinol-binding protein 1 (CRBP1) gene that was selected by using the microarray data. As CRBP1 regulates intracellular retinoic acid (vitamin A) homeostasis, which is involved in morphogenesis, and cellular proliferation and differentiation, the loss of CRBP1 could cause tumorigenesis in BC. We hypothesized that the inactivation of the CRBP1 gene through CpG methylation contributes to cell viability, including the migration and invasion activity of human BC cells. After the 5-aza-dC treatment, the mRNA and protein expression levels of CRBP1 markedly increased in all BOY and T24 BC cell lines. Combined bisulfite-restriction analysis and bisulfite DNA sequencing revealed that promoter CpG hypermethylation existed in 28 out of the 65 BCs (43%) and in none of the 16 normal bladder epithelia (NBEs). Conversely, CRBP1 mRNA expression in the BCs was significantly lower than that in the NBEs (0.63±0.11 vs. 4.92±0.80, p<0.0001). We found significant inhibition of cell growth (p<0.0001) and migration (p<0.0001) in the CRBP1 stable transfectants compared to the control cell line, in a cell proliferation and wound-healing assay, respectively. In conclusion, the aberrant CpG hypermethylation of the CRBP1 gene promoter could be involved in the development of BC. We demonstrate here for the first time that the CRBP1 gene could have a tumor suppressive function in BC.


Matsumoto M.,Kagoshima University | Kawakami K.,Kagoshima University | Enokida H.,Kagoshima University | Toki K.,Kagoshima University | And 6 more authors.
International Journal of Molecular Medicine | Year: 2010

We previously reported a simple technique that combines microarray data from clinical bladder cancer (BC) specimens with those from a BC cell line (BOY) treated with a pharmacologic demethylating agent (5-aza-dC). We focused on the human four-and-a-half LIM domains 1 (FHL1) gene which was selected on the basis of previous microarray data analysis. Because LIM domains provide protein-protein binding interfaces, FHL genes play an important role in cellular events, such as focal adhesion and differentiation, by interacting with the target protein as either a repressor or activator. We hypothesized that inactivation of the FHL1 gene through CpG methylation contributes to cell viability including migration and invasion activity of human BC. After 5-aza-dC treatment, the expression levels of FHL1 mRNA transcript markedly increased in all cell lines tested, as shown by real-time reverse transcription-polymerase chain reaction (RT-PCR). The methylation index of FHL1 in our samples was significantly higher in 70 BC specimens than in 10 normal bladder epithelium (NBE) specimens (63.9±25.5 and 0.3±0.2, respectively; p=0.0066). Conversely, FHL1 mRNA expression was significantly lower in the BC specimens than in the NBE ones (0.331±0.12 and 2.498±0.61, respectively; p=0.0011). In addition, significant inhibitions of wound healing (45.78±6.2, and 100±0, respectively; p=0.009) and of cell invasion (18.5±2.3 and 95.2±2.4, respectively; p=0.02) were observed in stable FHL1-transfected cells than in the control BC cells. In conclusion, we found that the mechanism of FHL1 down-regulation in BC is through CpG hypermethylation of the promoter region. FHL1 gene inactivation by CpG hypermethylation may thus contribute to migration and invasion activity of BC.


Chiyomaru T.,Kagoshima University | Enokida H.,Kagoshima University | Tatarano S.,Kagoshima University | Kawahara K.,Kawahara Nephro urology Clinic | And 6 more authors.
British Journal of Cancer | Year: 2010

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 (n46) was significantly higher than in non-invasive BC (n20) (P0.0055).Conclusion:Tumour suppressive miR-145 and miR-133a directly control oncogenic FSCN1 in BC. © 2010 Cancer Research UK All rights reserved.


Yamada Y.,Kagoshima University | Enokida H.,Kagoshima University | Kojima S.,Teikyo Chiba Medical Center | Kawakami K.,Kagoshima University | And 7 more authors.
Cancer Science | Year: 2011

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. © 2010 Japanese Cancer Association.


Tatarano S.,Kagoshima University | Chiyomaru T.,Kagoshima University | Kawakami K.,Kagoshima University | Enokida H.,Kagoshima University | And 7 more authors.
International Journal of Oncology | Year: 2011

Growing evidence suggests that microRNAs (miRNAs) are aberrantly expressed in many human cancers, and that they play significant roles in carcinogenesis and cancer progression. The identification of tumor suppressive miRNAs and their target genes could provide new insights into the mechanism of carcinogenesis. However, the genetic or epigenetic regulations of these miRNAs have not yet been fully elucidated in bladder cancer (BC). Chromosomal alterations of cancer cells give us important information for the identification of tumor suppressor genes. Our miRNA array-comparative genomic hybridization (CGH) analysis showed several miRNAs to be candidate tumor suppressors of BC. Our array-CGH analysis revealed that chromosome 4 was lost in all BC cell lines. We selected 19 miRNAs located on chromosome 4 and evaluated their expression levels in cancer cell lines as well as clinical samples. Gain-of-function analysis revealed that miR-218 inhibited BC cell proliferation, migration and invasion. Furthermore, flow cytometry analysis showed that it induced BC cell apoptosis. Genome-wide gene expression analysis showed that it targeted multiple oncogenes in BC. Our study is the first to demonstrate that miR-218 located on chrosomosme 4p15.31 is a tumor suppressive miRNA in BC. The identification of tumor suppressive miRNAs and their target genes on the basis of array-CGH analysis could provide new insights into the mechanisms of BC carcinogenesis.


Chiyomaru T.,Kagoshima University | Enokida H.,Kagoshima University | Kawakami K.,Kagoshima University | Tatarano S.,Kagoshima University | And 5 more authors.
Urologic Oncology: Seminars and Original Investigations | Year: 2012

Objective: 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. Methods: 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. Results: Real-time RT-PCR showed that LASP1 mRNA expression was higher in 76 clinical BC specimens than in 10 normal bladder epitheliums (P ≤ 0.05). Loss-of-function studies using si-LASP1-transfected BC cell lines demonstrated significant cell viability inhibition (P ≤ 0.0005), cell migration inhibition (P ≤ 0.0001), and a decrease in the number of invading cells (P ≤ 0.005) 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 (P ≤ 0.05), 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 (P ≤ 0.001). Conclusion: 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. © 2012 Elsevier Inc.

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