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 (p < 0.0001 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 5 0.0004). Spearman rank correlation analysis revealed significant inverse correlations between KRT7 mRNA expression and each downregulated miRNA (p < 0.0001 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. ' 2009 UICC

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.

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.

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-1 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.

BTG3/ANA/APRO4 has been reported to be a tumor suppressor gene in some malignancies. It constitutes important negative regulatory mechanism for Src-mediated signaling, a negative regulator of the cell cycle and inhibits transcription factor E2F1. We report that BTG3 is downregulated in renal cancer and that the mechanism of inactivation is through promoter hypermethylation. Quantitative real-time polymerase chain reaction (PCR) showed that BTG3 was downregulated in cancer tissues and cells. Genistein and 5-aza-2'-deoxycytidine (5Aza-C) induced BTG3 messenger RNA (mRNA) expression in A498, ACHN and HEK-293 renal cell carcinoma (RCC) cell lines. Bisulfite-modified PCR and DNA sequencing results showed complete methylation of BTG3 promoter in tumor samples and cancer cell lines. Genistein and 5Aza-C treatment significantly decreased promoter methylation, reactivating BTG3 expression. Chromatin immunoprecipitation assay revealed that genistein and 5Aza-C increased levels of acetylated histones 3, 4, 2H3K4, 3H3K4 and RNA polymerase II at the BTG3 promoter indicative of active histone modifications. Enzymatic assays showed genistein and 5Aza-C decreased DNA Methyltransferase, methyl-CpG-binding domain 2 activity and increased HAT activity. Cell cycle and 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide cell proliferation assays showed that genistein has antiproliferative effect on cancer cell growth through induction of cell cycle arrest. This is the first report to show that BTG3 is epigenetically silenced in RCC and can be reactivated by genistein-induced promoter demethylation and active histone modification. Genistein had similar effects to that of 5Aza-C, which is a potent demethylating agent with high toxicity and instability. Genistein being a natural, non-toxic, dietary isoflavone is effective in retarding the growth of RCC cells, making it a promising candidate for epigenetic therapy in renal carcinoma.