Hirofumi Yoshino

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, 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. (Cancer Sci 2011; 102: 522–529)

Tumour suppressors miR-1 and miR-133a target the oncogenic function of purine nucleoside phosphorylase (PNP) in prostate cancer

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.

Genistein Inhibits Prostate Cancer Cell Growth by Targeting miR-34a and Oncogenic HOTAIR

Objective Genistein is a soy isoflavone that has antitumor activity both in vitro and in vivo. It has been shown that genistein inhibits many type of cancers including prostate cancer (PCa) by regulating several cell signaling pathways and microRNAs (miRNAs). Recent studies suggest that the long non-coding RNAs (lncRNAs) are also involved in many cellular processes. At present there are no reports about the relationship between gensitein, miRNAs and lncRNAs. In this study, we focused on miRNAs, lncRNA that are regulated by genistein and investigated their functional role in PCa. Method Microarray (SurePrint G3 Human GE 8×60K) was used for expression profiling of genistein treated and control PCa cells (PC3 and DU145). Functional assay (cell proliferation, migration, invasion, apoptosis and cell cycle assays) were performed with the PCa cell lines, PC3 and DU145. Both in vitro and in vivo (nude mouse) models were used for growth assays. Luciferase reporter assays were used for binding of miR-34a to HOTAIR. Results LncRNA profiling showed that HOTAIR was highly regulated by genistein and its expression was higher in castration-resistant PCa cell lines than in normal prostate cells. Knockdown (siRNA) of HOTAIR decreased PCa cell proliferation, migration and invasion and induced apoptosis and cell cycle arrest. miR-34a was also up-regulated by genistein and may directly target HOTAIR in both PC3 and DU145 PCa cells. Conclusions Our results indicated that genistein inhibited PCa cell growth through down-regulation of oncogenic HOTAIR that is also targeted by tumor suppressor miR-34a. These findings enhance understanding of how genistein regulates lncRNA HOTAIR and miR-34a in PCa.

Aberrant expression of microRNAs in bladder cancer

MicroRNAs (miRNAs), a class of small noncoding RNAs, regulate protein-coding gene expression by repressing translation or cleaving RNA transcripts in a sequence-specific manner. A growing body of evidence suggests that miRNAs contribute to bladder cancer development, progression and metastasis. Genome-wide miRNA expression signatures have been used to rapidly and precisely identify aberrant miRNA expression in bladder cancer. Based on reports describing miRNA signatures, several downregulated and upregulated miRNAs have been discovered. Examination of the differential expression of miRNAs between clinical bladder cancer and normal bladder tissue has led to the elucidation of 11 miRNA expression signatures. miRNAs downregulated in bladder cancer, such as miR-145, miR-143 and miR125b, are known to be tumour suppressors, whereas upregulated miRNAs, such as miR-183, miR-96, miR17-5p and miR-20a are oncogenic. Several studies have demonstrated the potential of miRNAs for providing prognostic information. miR-145 is the most frequently downregulated miRNA in bladder cancer and has been shown to significantly inhibit proliferation, migration and invasion. Understanding the role of differentially expressed miRNAs, as well as their molecular targets, in bladder cancer will provide an effective and promising strategy for miRNA-based therapeutics for the treatment of bladder cancer.

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

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.

Tumor suppressive microRNA-375 regulates oncogene AEG-1 / MTDH in head and neck squamous cell carcinoma (HNSCC)

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.

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.

Tumor suppressive microRNAs ( miR-222 and miR-31 ) regulate molecular pathways based on microRNA expression signature in prostate cancer

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.