[show abstract][hide abstract] ABSTRACT: Current genome-wide microRNA (miRNA) expression signature analysis using deep sequencing technologies can drive the discovery of novel cancer pathways regulated by oncogenic and/or tumor suppressive miRNAs. We determined the genome-wide miRNA expression signature in bladder cancer (BC) by deep sequencing technology. A total of ten small RNA libraries were sequenced (five BCs and five samples of histologically normal bladder epithelia (NBE)), and 13,190,619 to 18,559,060 clean small RNA reads were obtained. A total of 933 known miRNAs and 17 new miRNA candidates were detected in this analysis. Among the known miRNAs, a total of 60 miRNAs were significantly downregulated in BC compared with NBE. We also found that several miRNAs, such as miR-1/133a, miR-206/133b, let-7c/miR-99a, miR-143/145 and miR-195/497, were located close together at five distinct loci and constituted clustered miRNAs. Among these clustered miRNAs, we focused on the miR-195/497 cluster because this clustered miRNA had not been analyzed in BC. Transfection of mature miR-195 or miR-497 in two BC cell lines (BOY and T24) significantly inhibited cancer cell proliferation, migration and invasion, suggesting that the miR-195/497 cluster functioned as tumor suppressors in BC. Regarding the genes targeted by the miR-195/497 cluster, the TargetScan algorithm showed that 6,730 genes were putative miR-195/497 targets, and 113 significantly enriched signaling pathways were identified in this analysis. The "Pathways in cancer" category was the most enriched, involving 104 candidate target genes. Gene expression data revealed that 27 of 104 candidate target genes were actually upregulated in BC clinical specimens. Luciferase reporter assays and Western blotting demonstrated that BIRC5 and WNT7A were directly targeted by miR-195/497. In conclusion, aberrant expression of clustered miRNAs was identified by deep sequencing, and downregulation of miR-195/497 contributed to BC progression and metastasis. Tumor suppressive miRNA-mediated cancer pathways provide new insights into the potential mechanisms of BC oncogenesis.
PLoS ONE 01/2014; 9(2):e84311. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Our recent studies of microRNA (miRNA) expression signatures demonstrated that microRNA-1291 (miR-1291) was significantly down-regulated in renal cell carcinoma (RCC) clinical specimens and was a putative tumor-suppressive miRNA in RCC. The aim of this study was to investigate the functional significance of miR-1291 in cancer cells and to identify novel miR-1291-mediated cancer pathways and target genes in RCC. The expression of miR-1291 was significantly down-regulated in RCC tissues compared with adjacent non-cancerous tissues. Restoration of mature miR-1291 in RCC cell lines (A498 and 786-O) revealed significant inhibition of cell proliferation, migration, and invasion, suggesting that miR-1291 functioned as a tumor suppressor. To identify miR-1291-mediated molecular pathways and targets, we used gene expression analysis (expression of RCC clinical specimens and miR-1291-transfected A498 cells) and in silico database analysis. Our data demonstrated that 79 signaling pathways were significantly regulated by tumor-suppressive miR-1291 in RCC cells. Moreover, solute career family 2 member 1 (SLC2A1) was a candidate target of miR-1291 regulation. The SLC2A1 gene provides instructions for producing glucose transporter protein type 1 (GLUT1). Luciferase reporter assays showed that miR-1291 directly regulated SLC2A1/GLUT1. In RCC clinical specimens, the expression of SLC2A1/GLUT1 mRNA was significantly higher in cancer tissues than in non-cancerous tissues. A significant inverse correlation was recognized between SLC2A1/GLUT1 and miR-1291 expression (r = -0.55, P < 0.0001). Loss of tumor-suppressive miR-1291 enhanced RCC cell proliferation, migration, and invasion through targeting SLC2A1/GLUT1. The identification of novel tumor-suppressive miR-1291-mediated molecular pathways and targets has provided new insights into RCC oncogenesis and metastasis. This article is protected by copyright. All rights reserved.
[show abstract][hide abstract] ABSTRACT: PURPOSE:: Our microRNA (miRNA) expression signature of renal cell carcinoma (RCC) revealed that miR-218expression was significantly reduced in cancer tissues, suggesting miR-218was a candidate tumor suppressor. The aim of this study was to investigate the functional significance of miR-218in cancer cells and to identify novel miR-218-mediated cancer pathways in RCC. MATERIALS AND METHODS:: Gain-of-function studies using mature miR-218were performed to investigate cell proliferation, migration and invasion in RCC cell lines (A498 and 786-O). To identify miR-218-mediated molecular pathways and responsible genes in RCC, we adopted gene expression analyses and in silico database analyses. Loss-of-function assays were performed to investigate the functional significance of miR-218target genes. RESULTS:: Restoration of mature miR-218significantly inhibited cell proliferation, migration and invasion in RCC cells. Gene expression studies and luciferase reporter assays showed that caveolin-2 ( CAV2) involved in focal adhesion pathway was directly regulated by miR-218. Silencing study of CAV2demonstrated significant inhibition of cell proliferation, migration and invasion. The mRNA and protein expression of CAV2 were significantly upregulated in RCC clinical specimens. CONCLUSIONS:: Loss of tumor suppressive miR-218 enhances cancer cell migration and invasion through dysregulation of the focal adhesion pathway, especially CAV2 as an oncogenic function in RCC. Tumor suppressive miRNA-mediated cancer pathways and responsible genes provide new insights into the potential mechanisms of RCC oncogenesis and metastasis.
The Journal of urology 02/2013; · 4.02 Impact Factor
[show abstract][hide abstract] ABSTRACT: Genistein has been shown to inhibit cancers both in vitro and in vivo, by altering the expression of several microRNAs (miRNAs). In this study, we focused on tumor suppressor miRNAs regulated by genistein and investigated their function in prostate cancer (PCa) and target pathways. Using miRNA microarray analysis and real-time RT-PCR we observed that miR-574-3p was significantly up-regulated in PCa cells treated with genistein compared with vehicle control. The expression of miR-574-3p was significantly lower in PCa cell lines and clinical PCa tissues compared with normal prostate cells (RWPE-1) and adjacent normal tissues. Low expression level of miR-574-3p was correlated with advanced tumor stage and higher Gleason score in PCa specimens. Re-expression of miR-574-3p in PCa cells significantly inhibited cell proliferation, migration and invasion in vitro and in vivo. miR-574-3p restoration induced apoptosis through reducing Bcl-xL and activating caspase-9 and caspase-3. Using GeneCodis software analysis, several pathways affected by miR-574-3p were identified, such as 'Pathways in cancer', 'Jak-STAT signaling pathway', and 'Wnt signaling pathway'. Luciferase reporter assays demonstrated that miR-574-3p directly binds to the 3' UTR of several target genes (such as RAC1, EGFR and EP300) that are components of 'Pathways in cancer'. Quantitative real-time PCR and Western analysis showed that the mRNA and protein expression levels of the three target genes in PCa cells were markedly down-regulated with miR-574-3p. Loss-of-function studies demonstrated that the three target genes significantly affect cell proliferation, migration and invasion in PCa cell lines. Our results show that genistein up-regulates tumor suppressor miR-574-3p expression targeting several cell signaling pathways. These findings enhance understanding of how genistein regulates with miRNA in PCa.
PLoS ONE 01/2013; 8(3):e58929. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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. Next, we 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 KEGG 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.
[show abstract][hide abstract] ABSTRACT: Recent our microRNA (miRNA) expression signature revealed that expression of microRNA-218 (miR-218) was reduced in cancer tissues, suggesting a candidate of tumor suppressor in head and neck squamous cell carcinoma (HNSCC). The aim of this study was to investigate the functional significance of miR-218 and its mediated moleculer pathways in HNSCC. Restoration of miR-218 in cancer cells led to significant inhibition of cell migration and invasion activities in HNSCC cell lines (FaDu and SAS). Genome-wide gene expression analysis of miR-218 transfectants and in silico database analysis showed that focal adhesion pathway was a promising candidate of miR-218 target pathways. The laminins are an important and biologically active part of the basal lamina, the function of that are various such as influencing cell differentiation, migration and adhesion as well as proliferation and cell survival. Interestingly, all components of laminin-332 (LAMA3, LAMB3 and LAMC2) are listed on the candidate genes in focal adhesion pathway. Furthermore, we focused on LAMB3 which has a miR-218 target site and gene expression studies and luciferase reporter assays showed that LAMB3 was directly regulated by miR-218. Silencing study of LAMB3 demonstrated significant inhibition of cell migration and invasion. In clinical specimens with HNSCC, the expression levels of laminin-332 were significantly upregulated in cancer tissues compared to adjacent non-cancerous tissues. Our analysis data showed that tumor suppressive miR-218 contributes to cancer cell migration and invasion through regulating focal adhesion pathway, especially laminin-332. Tumor suppressive miRNA-mediated novel cancer pathways provide new insights into the potential mechanisms of HNSCC oncogenesis.
[show abstract][hide abstract] ABSTRACT: 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.
International Journal of Oncology 07/2012; 41(3):805-17. · 2.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: Our expression signatures of human cancers including head and neck squamous cell carcinoma (HNSCC) demonstrated that downregulation of microRNA-133a (miR-133a) were frequently observed in cancer cells. The restoration of miR-133a in cancer cells revealed that it functions as a tumor suppressor. In this study, we investigated the novel molecular targets of miR-133a in HNSCC cancer cells and its oncogenic function, especially as it contributes to cancer cell migration and invasion. The genome-wide gene expression analysis and bioinformatics study showed that actin-related protein 2/3 complex subunit 5 (ARPC5) is a candidate target of miR-133a. Furthermore, luciferase reporter assay demonstrated that ARPC5 is directly regulated by miR-133a. Silencing of ARPC5 revealed significant inhibition of cell migration and invasion in HNSCC cell lines, SAS, HSC3 and IMC-3. In HSC3 cells, restoration of miR-133a or silencing ARPC5 led to a reorganization of the actin cytoskeleton and a subsequent change in cell morphology to a round, bleb-like shape. The expression levels of ARPC5 were significantly higher in HNSCC tissues than in non-cancer tissues. Immunohistochemistry showed that the levels of ARPC5 expression were significantly higher in invasive cancer cells. ARPC5 contributed to cancer cell migration and invasion in HNSCC and this gene was directly regulated by miR-133a. Our analysis of novel tumor-suppressive miR‑133a-mediated cancer pathways provides new insights into the potential mechanisms of HNSCC oncogenesis.
International Journal of Oncology 02/2012; 40(6):1770-8. · 2.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: Our expression signatures of human cancer including bladder cancer (BC) revealed that the expression of microRNA-1 (miR-1) and microRNA-133a (miR-133a) is significantly reduced in cancer cells. In the human genome, miR-1 and miR-133a are located on the same chromosomal region (miR-1-2 and miR-133a-1 on 18q11.2, and miR-1-1 and miR-133a-2 on 20q13.33) called cluster. In this study, we identified the novel molecular targets commonly regulated by miR-1 and miR-133a in BC. Genome-wide molecular target search and luciferase reporter assays showed that prothymosin-α (PTMA) and purine nucleoside phosphorylase (PNP) are directly regulated by miR-1 and miR-133a. Silencing of these two genes significantly inhibited cell proliferation and invasion, and increased apoptosis in BC cells. Immunohistochemistry showed that PTMA expression levels were significantly higher in BC compared to normal bladder epitheliums. PTMA and PNP were identified as new target genes regulated by the miR-1 and miR-133a cluster in BC. These genes may function as oncogenes contributing to cell proliferation and invasion in BC. Tumor suppressive miR-1 and miR-133a-mediated novel molecular targets may provide new insights into the potential mechanisms of BC oncogenesis.
International Journal of Oncology 02/2012; 40(6):1821-30. · 2.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: Our previous studies suggested that microRNA (miR)-574-3p is a candidate tumor suppressor microRNA (miRNA) in human bladder cancer (BC). Among 17 down-regulated miRNAs, miR-574-3p is located on chromosome 4p14 where we had identified a chromosomal loss region by array-CGH in BC cell lines. MiR-574-3p expression was down-regulated in BC cell lines. Gain-of-function analysis revealed that cell proliferation, migration and invasion were significantly inhibited in miR‑574‑3p-transfected BC cell lines. Flow cytometry analysis showed that cell apoptosis was induced in miR-574-3p transfectants. Oligo microarray analysis suggested that the mesoderm development candidate 1 (MESDC1) gene was a target gene in miR-574-3p transfectants. Luciferase assays revealed that miR‑574‑3p was directly bound to MESDC1 mRNA. MESDC1 is predicted to be a novel actin-binding protein located on chromosome 15q13. Although the gene is conserved among many species, its functional role is still unknown in both human malignancies and normal tissues. Loss-of-function studies demonstrated that cell proliferation, migration and invasion were significantly inhibited in si-MESDC1-transfected BC cell lines. Flow cytometry analysis showed that apoptosis was induced in si-MESDC1 transfectants. We are the first to demonstrate that miR-574-3p is a miRNA with tumor suppressor function and that MESDC1 (which has a potential oncogenic function in BC) may be targeted by miR-574-3p.
International Journal of Oncology 12/2011; 40(4):951-9. · 2.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Biochemical and Biophysical Research Communications 12/2011; 417(1):588-93. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
International Journal of Oncology 07/2011; 39(1):13-21. · 2.66 Impact Factor