Hiroko Mataki

Tumour-suppressive microRNA-144-5p directly targets CCNE1 / 2 as potential prognostic markers in bladder cancer

Background:Analysis of a microRNA (miRNA) expression signature of bladder cancer (BC) by deep-sequencing revealed that clustered miRNAs microRNA (miR)-451a, miR-144-3p, and miR-144-5p were significantly downregulated in BC tissues. We hypothesised that these miRNAs function as tumour suppressors in BC. The aim of this study was to investigate the functional roles of these miRNAs and their modulation of cancer networks in BC cells.Methods:The functional studies of BC cells were performed using transfection of mature miRNAs. Genome-wide gene expression analysis, in silico analysis, and dual-luciferase reporter assays were applied to identify miRNA targets. The association between miR-144-5p levels and expression of the target genes was determined, and overall patient survival as a function of target gene expression was estimated by the Kaplan–Meier method.Results:Gain-of-function studies showed that miR-144-5p significantly inhibited cell proliferation by BC cells. Four cell cycle-related genes (CCNE1, CCNE2, CDC25A, and PKMYT1) were identified as direct targets of miR-144-5p. The patients with high CCNE1 or CCNE2 expression had lower overall survival probabilities than those with low expression (P=0.025 and P=0.032).Conclusion:miR-144-5p functions as tumour suppressor in BC cells. CCNE1 and CCNE2 were directly regulated by miR-144-5p and might be good prognostic markers for survival of BC patients.

Dual regulation of receptor tyrosine kinase genes EGFR and c-Met by the tumor-suppressive microRNA-23b/27b cluster in bladder cancer

Recent clinical trials of chemotherapeutics for advanced bladder cancer (BC) have shown limited benefits. Therefore, new prognostic markers and more effective treatment strategies are required. One approach to achieve these goals is through the analysis of RNA networks. Our recent studies of microRNA (miRNA) expression signatures revealed that the microRNA-23b/27b (miR-23b/27b) cluster is frequently downregulated in various types of human cancers. However, the functional role of the miR-23b/27b cluster in BC cells is still unknown. Thus, the aim of the present study was to investigate the functional significance of the miR-23b/27b cluster and its regulated molecular targets, with an emphasis on its contributions to BC oncogenesis and metastasis. The expression levels of the miR-23b/27b cluster were significantly reduced in BC clinical specimens. Restoration of mature miR-23b or miR-27b miRNAs significantly inhibited cancer cell migration and invasion, suggesting that these clustered miRNAs function as tumor suppressors. Gene expression data and in silico analysis demonstrated that the genes coding for the epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (c-Met) were potential targets of the miR-23b/27b cluster. Luciferase reporter assays and western blotting demonstrated that EGFR and c-Met receptor trypsine kinases were directly regulated by these clustered miRNAs. We conclude that the decreased expression of the tumor-suppressive miR-23b/27b cluster enhanced cancer cell proliferation, migration and invasion in BC through direct regulation of EGFR and c-Met signaling pathways. Our data on RNA networks regulated by tumor-suppressive miR-23b/27b provide new insights into the potential mechanisms of BC oncogenesis and metastasis.

Tumour-suppressive microRNA-24-1 inhibits cancer cell proliferation through targeting FOXM1 in bladder cancer.

Here, we found that microRNA‐24‐1 (miR‐24‐1) is significantly reduced in bladder cancer (BC) tissues, suggesting that it functions as a tumour suppressor. Restoration of mature miR‐24‐1 inhibits cancer cell proliferation and induces apoptosis. Forkhead box protein M1 (FOXM1) is a direct target gene of miR‐24‐1, as shown by genome‐wide gene expression analysis and luciferase reporter assay. Overexpressed FOXM1 is confirmed in BC clinical specimens, and silencing of FOXM1 induces apoptosis in cancer cell lines. Our data demonstrate that the miR‐24‐1 – FOXM1 axis contributes to cancer cell proliferation in BC, and elucidation of downstream signalling will provide new insights into the molecular mechanisms of BC oncogenesis.

Downregulation of the microRNA-1/133a cluster enhances cancer cell migration and invasion in lung-squamous cell carcinoma via regulation of Coronin1C

Lung cancer is clearly the primary cause of cancer-related deaths worldwide. Recent molecular-targeted strategy has contributed to improvement of the curative effect of adenocarcinoma of the lung. However, such current treatment has not been developed for squamous cell carcinoma (SCC) of the disease. The new genome-wide RNA analysis of lung-SCC may provide new avenues for research and the development of the disease. Our recent microRNA (miRNA) expression signatures of lung-SCC revealed that clustered miRNAs miR-1/133a were significantly reduced in cancer tissues. Here, we found that restoration of both mature miR-1 and miR-133a significantly inhibited cancer cell proliferation, migration and invasion. Coronin-1C (CORO1C) was a common target gene of the miR-1/133a cluster, as shown by the genome-wide gene expression analysis and the luciferase reporter assay. Silencing of CORO1C gene expression inhibited cancer cell proliferation, migration and invasion. Furthermore, CORO1C-regulated molecular pathways were categorized by using si-CORO1C transfectants. Further analysis of novel cancer signaling pathways modulated by the tumor-suppressive cluster miR-1/133a will provide insights into the molecular mechanisms of lung-SCC oncogenesis and metastasis.

Dual-strand tumor-suppressor microRNA-145 ( miR-145-5p and miR-145-3p ) coordinately targeted MTDH in lung squamous cell carcinoma

Patients with lung adenocarcinoma may benefit from recently developed molecular targeted therapies. However, analogous advanced treatments are not available for patients with lung squamous cell carcinoma (lung SCC). The survival rate of patients with the advanced stage of lung SCC remains poor. Exploration of novel lung SCC oncogenic pathways might lead to new treatment protocols for the disease. Based on this concept, we have identified microRNA- (miRNA) mediated oncogenic pathways in lung SCC. It is well known that miR-145-5p (the guide strand) functions as a tumor suppressor in several types of cancer. However, the impact of miR-145-3p (the passenger strand) on cancer cells is still ambiguous. Expression levels of miR-145-5p and miR-145-3p were markedly reduced in cancer tissues, and ectopic expression of these miRNAs inhibited cancer cell aggressiveness, suggesting that both miR-145-3p as well as miR-145-5p acted as antitumor miRNAs. We identified seven putative target genes (MTDH, EPN3, TPD52, CYP27B1, LMAN1, STAT1 and TXNDC12) that were coordinately regulated by miR-145-5p and miR-145-3p in lung SCC. Among the seven genes, we found that metadherin (MTDH) was a direct target of these miRNAs. Kaplan–Meier survival curves showed that high expression of MTDH predicted reduced survival of lung SCC patients. We investigated pathways downstream from MTDH by using genome-wide gene expression analysis. Our data showed that several anti-apoptosis and pro-proliferation genes were involved in pathways downstream from MTDH in lung SCC. Taken together, both strands of miR-145, miR-145-5p and miR-145-3p are functional and play pivotal roles as antitumor miRNAs in lung SCC.

Tumor-suppressive microRNA-29 family inhibits cancer cell migration and invasion directly targeting LOXL2 in lung squamous cell carcinoma

Lung cancer remains the most frequent cause of cancer-related death in developed countries. A recent molecular-targeted strategy has contributed to improvement of the remarkable effect of adenocarcinoma of the lung. However, such treatment has not been developed for squamous cell carcinoma (SCC) of the disease. Our recent studies of microRNA (miRNA) expression signatures of human cancers showed that the microRNA-29 family (miR-29a, miR-29b and miR-29c) significantly reduced cancer tissues compared to normal tissues. These findings suggest that miR-29s act as tumor-suppressors by targeting several oncogenic genes. The aim of the study was to investigate the functional significance of miR-29s in lung SCC and to identify miR-29s modulating molecular targets in lung SCC cells. Restoration of all mature members of the miR-29s inhibited cancer cell migration and invasion. Gene expression data combined in silico analysis and luciferase reporter assays demonstrated that the lysyl oxidase-like 2 (LOXL2) gene was a direct regulator of tumor-suppressive miR-29s. Moreover, overexpressed LOXL2 was confirmed in lung SCC clinical specimens, and silencing of LOXL2 inhibited cancer cell migration and invasion in lung SCC cell lines. Our present data suggested that loss of tumor-suppressive miR-29s enhanced cancer cell invasion in lung SCC through direct regulation of oncogenic LOXL2. Elucidation of the novel lung SCC molecular pathways and targets regulated by tumor-suppressive miR-29s will provide new insights into the potential mechanisms of oncogenesis and metastasis of the disease.

MicroRNAs in non-small cell lung cancer and idiopathic pulmonary fibrosis

In spite of advances in the diagnosis and current molecular target therapies of lung cancer, this disease remains the most common cause of cancer-related death worldwide. Approximately 80% of lung cancers is non-small cell lung cancer (NSCLC), and 5-year survival rate of the disease is ~20%. On the other hand, idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease of unknown etiology. IPF is refractory to treatment and has a very low survival rate. Moreover, IPF is frequently associated with lung cancer. However, the common mechanisms shared by these two diseases remain poorly understood. In the post-genome sequence era, the discovery of noncoding RNAs, particularly microRNAs (miRNAs), has had a major impact on most biomedical fields, and these small molecules have been shown to contribute to the pathogenesis of NSCLC and IPF. Investigation of novel RNA networks mediated by miRNAs has improved our understanding of the molecular mechanisms of these diseases. This review summarizes our current knowledge on aberrantly expressed miRNAs regulating NSCLC and IPF based on miRNA expression signatures.

Dual-receptor (EGFR and c-MET) inhibition by tumor-suppressive miR-1 and miR-206 in head and neck squamous cell carcinoma.

Our studies of microRNA (miRNA) expression signatures have shown that microRNA-1 (miR-1) and microRNA-206 (miR-206) were downregulated in head and neck squamous cell carcinoma (HNSCC) clinical specimens. The seed sequences of these miRNAs are identical, suggesting that the identification of the molecular targets regulated by miR-1 and miR-206 will provide new insights into novel mechanisms of HNSCC pathogenesis. Our present data showed that restoration of miR-1 and miR-206 significantly inhibited HNSCC cells’ aggressiveness. A combination of gene expression data and in silico analysis revealed that several pathways (‘pathway in cancer’, ‘focal adhesion pathway’, ‘MAPK signaling pathway’, ‘regulation of actin cytoskeleton pathway’ and ‘ECM–receptor interaction pathway’) were regulated by miR-1 and miR-206. Among them, we found that two growth factor receptors, epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (c-MET), were directly regulated by both miR-1 and miR-206 in HNSCC cells. Also, downstream oncogenic signaling of these receptors was reduced by restoration of miR-1 or miR-206 expression. Moreover, overexpression of EGFR and c-MET was observed in HNSCC clinical specimens. The identification of targets modulated by tumor-suppressive miR-1 and miR-206 may lead to a better understanding of molecular pathogenesis of HNSCC.

Regulation of LOXL2 and SERPINH1 by antitumor microRNA-29a in lung cancer with idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive lung disease that is refractory to treatment and carries a high mortality rate. IPF is frequently associated with lung cancer. Identification of molecular targets involved in both diseases may elucidate novel molecular mechanisms contributing to their pathology. Recent studies of microRNA (miRNA) expression signatures showed that microRNA-29a (miR-29a) was downregulated in IPF and lung cancer. The aim of this study was to investigate the functional significance of miR-29a in lung cancer cells (A549 and EBC-1) and lung fibroblasts (MRC-5) and to identify molecular targets modulated by miR-29a in these cells. We confirmed the downregulation of miR-29a in clinical specimens of IPF and lung cancer. Restoration of miR-29a suppressed cancer cell aggressiveness and fibroblast migration. A combination of gene expression data and in silico analysis showed that a total of 24 genes were putative targets of miR-29a. Among them, lysyl oxidase-like 2 (LOXL2) and serpin peptidase inhibitor clade H, member 1 (SERPINH1) were direct targets of miR-29a by luciferase reporter assays. The functions of LOXL2 and SERPINH1 contribute significantly to collagen biosynthesis. Overexpression of LOXL2 and SERPINH1 was observed in clinical specimens of lung cancer and fibrotic lesions. Downregulation of miR-29a caused overexpression of LOXL2 and SERPINH1 in lung cancer and IPF, suggesting that these genes are involved in the pathogenesis of these two diseases.

Regulation of TPD52 by antitumor microRNA-218 suppresses cancer cell migration and invasion in lung squamous cell carcinoma

The development of targeted molecular therapies has greatly benefited patients with lung adenocarcinomas. In contrast, these treatments have had little benefit in the management of lung squamous cell carcinoma (lung SCC). Therefore, new treatment options based on current genomic approaches are needed for lung SCC. Aberrant microRNA (miRNA) expression has been shown to promote lung cancer development and aggressiveness. Downregulation of microRNA-218 (miR-218) was frequently observed in our miRNA expression signatures of cancers, and previous studies have shown an antitumor function of miR-218 in several types of cancers. However, the impact of miR-218 on lung SCC is still ambiguous. The present study investigated the antitumor roles of miR-218 in lung SCC to identify the target genes regulated by this miRNA. Ectopic expression of miR-218 greatly inhibited cancer cell migration and invasion in the lung SCC cell lines EBC-1 and SK-MES-1. Through a combination of in silico analysis and gene expression data searching, tumor protein D52 (TPD52) was selected as a putative target of miR-218 regulation. Moreover, direct binding of miR-218 to the 3′-UTR of TPD52 was observed by dual luciferase reporter assay. Overexpression of TPD52 was observed in lung SCC clinical specimens, and knockdown of TPD52 significantly suppressed cancer cell migration and invasion in lung SCC cell lines. Furthermore, the downstream pathways mediated by TPD52 involved critical regulators of genomic stability and mitotic checkpoint genes. Taken together, our data showed that downregulation of miR-218 enhances overexpression of TPD52 in lung SCC cells, promoting cancer cell aggressiveness. Identification of tumor-suppressive miRNA-mediated RNA networks of lung SCC will provide new insights into the potential mechanisms of the molecular pathogenesis of the disease.