Nijiro Nohata

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.

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.

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.

miR-489 is a tumour-suppressive miRNA target PTPN11 in hypopharyngeal squamous cell carcinoma (HSCC)

Background:Hypopharyngeal squamous cell carcinoma (HSCC) is an aggressive malignancy with one of the worst prognoses among all head and neck cancers. Greater understanding of the pertinent molecular oncogenic pathways could help improve diagnosis, therapy, and prevention of this disease. The aim of this study was to identify tumour-suppressive microRNAs (miRNAs), based on miRNA expression signatures from clinical HSCC specimens, and to predict their biological target genes.Methods:Expression levels of 365 human mature miRNAs from 10 HSCC clinical samples were screened using stem-loop real-time quantitative PCR. Downregulated miRNAs were used in cell proliferation assays to identify a tumour-suppressive miRNA. Genome-wide gene expression analyses were then performed to identify the target genes of the tumour-suppressive miRNA.Results:Expression analysis identified 11 upregulated and 31 downregulated miRNAs. Gain-of-function analysis of the downregulated miRNAs revealed that miR-489 inhibited cell growth in all head and neck cancer cell lines examined. The gene PTPN11 coding for a cytoplasmic protein tyrosine phosphatase containing two Src Homology 2 domains was identified as a miR-489-targeted gene. Knockdown of PTPN11 resulted in the inhibition of cell proliferation in head and neck SCC cells.Conclusion:Identification of the tumour-suppressive miRNA miR-489 and its target, PTPN11, might provide new insights into the underlying molecular mechanisms of HSCC.

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.

Tumor suppressive microRNA-133a regulates novel molecular networks in lung squamous cell carcinoma

Analysis of the microRNA (miRNA) expression signature of lung squamous cell carcinoma (lung-SCC) revealed that the expression levels of miR-133a were significantly reduced in cancer tissues compared with normal tissues. In this study, we focused on the functional significance of miR-133a in cancer cell lines derived from lung-SCC and the identification of miR-133a-regulated novel cancer networks in lung-SCC. Restoration of miR-133a expression in PC10 and H157 cell lines resulted in significant inhibition of cell proliferation, suggesting that miR-133a functions as a tumor suppressor. We used genome-wide gene expression analysis to identify the molecular targets of miR-133a regulation. Gene expression data and web-based searching revealed several candidate genes, including transgelin 2 (TAGLN2), actin-related protein2/3 complex, subunit 5, 16kDa (ARPC5), LAG1 homolog, ceramide synthase 2 (LASS2) and glutathione S-transferase pi 1 (GSTP1). ARPC5 and GSTP1 likely represent bona fide targets as their expression is elevated in lung-SCC clinical specimens. Furthermore, transient transfection of miR-133a, repressed ARPC5 and GSTP1 mRNA and protein levels. As cell proliferation was significantly inhibited in lung-SCC cells following RNAi knock down of either gene, ARPC5 and GSTP1 may function as oncogenes in the development of lung-SCC. The identification of a tumor suppressive miRNA and the novel cancer pathways it regulates could provide new insights into potential molecular mechanisms of lung-SCC carcinogenesis.

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.

The MicroRNA Expression Signature of Bladder Cancer by Deep Sequencing: The Functional Significance of the miR-195/497 Cluster

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.

Tumour suppressive microRNA-874 regulates novel cancer networks in maxillary sinus squamous cell carcinoma

Background:On the basis of the microRNA (miRNA) expression signature of maxillary sinus squamous cell carcinoma (MSSCC), we found that miR-874 was significantly reduced in cancer cells. We focused on the functional significance of miR-874 in cancer cells and identification of miR-874-regulated novel cancer networks in MSSCC.Methods:We used PCR-based methods to investigate the downregulated miRNAs in clinical specimens of MSSCC. Our signature analyses identified 23 miRNAs that were significantly reduced in cancer cells, such as miR-874, miR-133a, miR-375, miR-204, and miR-1. We focused on miR-874 as the most downregulated novel miRNA in our analysis.Results:We found potential tumour suppressive functions such as inhibition of cancer cell proliferation and invasion. A molecular target search of miR-874 revealed that PPP1CA was directly regulated by miR-874. Overexpression of PPP1CA was observed in MSSCC clinical specimens. Silencing of the PPP1CA gene significantly inhibited cancer cell proliferation and invasion.Conclusion:The downregulation of miR-874 was a frequent event in MSSCC, which suggests that miR-874 functions as a tumour suppressive miRNA, directly regulating PPP1CA that has a potential role of an oncogene. The identification of novel miR-874-regulated cancer pathways could provide new insights into potential molecular mechanisms of MSSCC oncogenesis.

Identification of novel molecular targets regulated by tumor suppressive miR-1/miR-133a in maxillary sinus squamous cell carcinoma

Based on our microRNA (miRNA) expression signature analysis of maxillary sinus squamous cell carcinoma (MSSCC), we found that miR-1 and miR-133a were significantly reduced in tumor tissues. Quantitative real-time RT-PCR revealed that the expression levels of miR-1 and miR-133a were significantly downregulated in clinical MSSCC tumor tissues compared with normal tissues. We focused on the functional significance of miR-1 and miR-133a in cancer cells and identification of the novel cancer networks regulated by these miRNAs in MSSCC. Restoration of downregulated miRNAs (miR-1 or miR-133a) in cancer cells revealed that both miRNAs significantly inhibited cancer cell proliferation and induced cell apoptosis. Molecular target identification of these miRNAs showed that transgelin 2 (TAGLN2) and purine nucleoside phosphorylase (PNP) were regulated by miR-1 and miR-133a. Both TAGLN2 and PNP mRNA expression levels were significantly upregulated in clinical MSSCC tumor tissues. Silencing studies of target genes demonstrated that both genes inhibited cancer cell proliferation. The identification of novel miR-1/miR-133a-regulated cancer pathways could provide new insights into potential molecular mechanisms of MSSCC oncogenesis.