Shuichi Tatarano

miR-145 and miR-133a function as tumour suppressors and directly regulate FSCN1 expression in bladder cancer

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.

The tumour-suppressive function of miR-1 and miR-133a targeting TAGLN2 in bladder cancer

Background:On the base of the microRNA (miRNA) expression signature of bladder cancer (BC), we found that miR-1 and miR-133a were significantly downregulated in BC. In this study, we focussed on the functional significance of miR-1 and miR-133a in BC cell lines and identified a molecular network of these miRNAs.Methods and results:We investigated the miRNA expression signature of BC clinical specimens and identified several downregulated miRNAs (miR-133a, miR-204, miR-1, miR-139-5p, and miR-370). MiR-1 and miR-133a showed potential role of tumour suppressors by functional analyses of BC cells such as cell proliferation, apoptosis, migration, and invasion assays. Molecular target searches of these miRNAs showed that transgelin 2 (TAGLN2) was directly regulated by both miR-1 and miR-133a. Silencing of TAGLN2 study demonstrated significant inhibitions of cell proliferation and increase of apoptosis in BC cell lines. The immunohistochemistry showed a positive correlation between TAGLN2 expression and tumour grade in clinical BC specimens.Conclusions:The downregulation of miR-1 and miR-133a was a frequent event in BC, and these miRNAs were recognised as tumour suppressive. TAGLN2 may be a target of both miRNAs and had a potential oncogenic function. Therefore, novel molecular networks provided by miRNAs may provide new insights into the underlying molecular mechanisms of BC.

MiR‐96 and miR‐183 detection in urine serve as potential tumor markers of urothelial carcinoma: correlation with stage and grade, and comparison with urinary cytology

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, Pu2003=u20030.0059; and miR‐183, Pu2003=u20030.0044). 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, Pu2003=u20030.0057; and miR‐183, Pu2003=u20030.0036) and pathological stage (miR‐96, Pu2003=u20030.0332; and miR‐183, Pu2003=u20030.0117). The expression levels of the microRNA were significantly lower in urine collected after surgery (miR‐96, Pu2003=u20030.0241; and miR‐183, Pu2003=u20030.0045). 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. (Cancer Sci 2011; 102: 522–529)

The functional significance of miR-1 and miR-133a in renal cell carcinoma

PURPOSEnThe 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.nnnMETHODSnWe 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.nnnRESULTSnThe 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.nnnCONCLUSIONSnOur 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.

Functional role of LASP1 in cell viability and its regulation by microRNAs in bladder cancer.

OBJECTIVEnOur 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.nnnMETHODSnThe 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.nnnRESULTSnReal-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).nnnCONCLUSIONnOur 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.

miR-218 on the genomic loss region of chromosome 4p15.31 functions as a tumor suppressor in bladder cancer

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.

Tumor‐suppressive microRNA‐143/145 cluster targets hexokinase‐2 in renal cell carcinoma

Our recent studies of microRNA (miRNA) expression signatures have indicated that the miR‐143/145 cluster is significantly downregulated in several types of cancer and represents a putative tumor‐suppressive miRNA in human cancers. The aim of this study was to investigate the functional significance of the miR‐143/145 cluster in cancer cells and to identify novel molecular targets of the miR‐143/145 cluster in renal cell carcinoma (RCC). The expression levels of miR‐143 and miR‐145 were significantly downregulated in RCC tissues compared with adjacent non‐cancerous tissues. A significant positive correlation was recognized between miR‐143 and miR‐145 expression. Restoration of mature miR‐143 or miR‐145 in 786‐O and A498 RCC cells revealed that both mature miRNAs significantly inhibited cancer cell proliferation and invasion, suggesting that the miR‐143/145 cluster functioned as a tumor suppressor in RCC. Gene expression data and in silico database analysis showed that the hexokinase‐2 (HK2) gene, which encodes a glycolytic enzyme crucial for the Warburg effect in cancer cells, was a candidate target of the miR‐143/145 cluster. Luciferase reporter assays showed that both miR‐143 and miR‐145 directly regulated HK2. In RCC clinical specimens, the expression of HK2 was significantly higher in cancer tissues than in non‐cancerous tissues. Silencing HK2 suppressed RCC cell proliferation and invasion, suggesting that HK2 has oncogenic functions in RCC. Thus, our data showed that loss of the tumor‐suppressive miR‐143/145 cluster enhanced RCC cell proliferation and invasion through targeting HK2.

MiR-133a induces apoptosis through direct regulation of GSTP1 in bladder cancer cell lines

OBJECTIVEnWe 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.nnnMETHODSnTo 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.nnnRESULTSnMiR-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 (P < 0.005). GSTP1 mRNA expression levels in 43 clinical BCs were significantly higher than those in eight normal bladder epitheliums (P = 0.0277).nnnCONCLUSIONnOur 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.

MicroRNA-218 Inhibits Cell Migration and Invasion in Renal Cell Carcinoma through Targeting Caveolin-2 Involved in Focal Adhesion Pathway

PURPOSEnOur microRNA expression signature of renal cell carcinoma revealed that miR-218 expression was significantly decreased in cancer tissues, suggesting that miR-218 is a candidate tumor suppressor. We investigated the functional significance of miR-218 in cancer cells and identified what are to our knowledge novel miR-218 mediated cancer pathways in renal cell carcinoma.nnnMATERIALS AND METHODSnGain of function studies using mature miR-218 were performed to investigate cell proliferation, migration and invasion in the A498 and 786-O renal cell carcinoma cell lines. To identify miR-218 mediated molecular pathways and responsible genes in renal cell carcinoma, we used gene expression and in silico database analyses. Loss of function assays were performed to investigate the functional significance of miR-218 target genes.nnnRESULTSnRestoration of mature miR-218 significantly inhibited RCC cell proliferation, migration and invasion. Gene expression studies and luciferase reporter assays showed that CAV2 involved in the focal adhesion pathway was directly regulated by miR-218. A silencing study of CAV2 revealed significant inhibition of cell proliferation, migration and invasion. CAV2 mRNA and protein expression was significantly up-regulated in renal cell carcinoma clinical specimens.nnnCONCLUSIONSnLoss 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 renal cell carcinoma. Tumor suppressive microRNA mediated cancer pathways and responsible genes provide new insights into the potential mechanisms of renal cell carcinoma oncogenesis and metastasis.

Epithelial–mesenchymal transition-related microRNA-200s regulate molecular targets and pathways in renal cell carcinoma

Our recent studies of microRNA (miRNA) expression signatures demonstrated that the epithelial–mesenchymal transition (EMT)-related microRNA-200 family (miR-200s: miR-200a/b/c, miR-141 and miR-429) were significantly downregulated in renal cell carcinoma (RCC) and putative tumor-suppressive miRNAs in RCC. In this study, our aim was to investigate the functional significance of the miR-200s in cancer cells and to identify novel miR-200s-regulated molecular targets and pathways in RCC. Expression levels of all the miR-200s members were significantly downregulated in human RCC tissues compared with normal renal tissues. Restoration of mature miR-200s in RCC cell line resulted in significant inhibition of cell proliferation and migration, suggesting that miR-200s function as tumor suppressors in RCC. Furthermore, we utilized gene expression analysis and in silico database analysis to identify miR-200s-regulated molecular targets and pathways in RCC. The miR-200s was categorized into two groups, according to their seed sequences, miR-200b/c/429 and miR-200a/141. Our data demonstrated that the ‘Focal adhesion’ and ‘ErbB signaling’ pathways were significantly regulated by miR-200b/c/429 and miR-200a/141, respectively. The identification of novel tumor-suppressive miR-200s-regulated molecular targets and pathways has provided new insights into RCC oncogenesis and metastasis.