Lisa Fujimura

miR-145, miR-133a and miR-133b: tumor-suppressive miRNAs target FSCN1 in esophageal squamous cell carcinoma

MicroRNAs (miRNAs), noncoding RNAs 21–25 nucleotides in length, regulate gene expression primarily at the posttranscriptional level. Growing evidence suggests that miRNAs are aberrantly expressed in many human cancers, and that they play significant roles in carcinogenesis and cancer progression. A search for miRNAs with a tumor‐suppressive function in esophageal squamous cell carcinoma (ESCC) was performed using the miRNA expression signatures obtained from ESCC clinical specimens. A subset of 15 miRNAs was significantly downregulated in ESCC. A comparison of miRNA signatures from ESCC and our previous report identified 4 miRNAs that are downregulated in common (miR‐145, miR‐30a‐3p, miR‐133a and miR‐133b), suggesting that these miRNAs are candidate tumor suppressors. Gain‐of‐function analysis revealed that 3 transfectants (miR‐145, miR‐133a and miR‐133b) inhibit cell proliferation and cell invasion in ESCC cells. These miRNAs (miR‐145, miR‐133a and miR‐133b), which have conserved sequences in the 3′UTR of FSCN1 (actin‐binding protein, Fascin homolog 1), inhibited FSCN1 expression. The signal from a luciferase reporter assay was significantly decreased at 2 miR‐145 target sites and 1 miR‐133a/b site, suggesting both miRNAs directly regulate FSCN1. An FSCN1 loss‐of‐function assay found significant cell growth and invasion inhibition, implying an FSCN1 is associated with ESCC carcinogenesis. The identification of tumor‐suppressive miRNAs, miR‐145, miR‐133a and miR‐133b, directly control oncogenic FSCN1 gene. These signal pathways of ESCC could provide new insights into potential mechanisms of ESCC carcinogenesis.

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.

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.

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.

Kinetics and protective role of autophagy in a mouse cecal ligation and puncture-induced sepsis

IntroductionIt is not well understood whether the process of autophagy is accelerated or blocked in sepsis, and whether it is beneficial or harmful to the immune defense mechanism over a time course during sepsis. Our aim was to determine both the kinetics and the role of autophagy in sepsis.MethodsWe examined autophagosome and autolysosome formation in a cecal ligation and puncture (CLP) mouse model of sepsis (in C57BL/6N mice and GFP-LC3 transgenic mice), using western blotting, immunofluorescence, and electron microscopy. We also investigated the effect of chloroquine inhibition of autophagy on these processes.ResultsAutophagy, as demonstrated by increased LC3-II/LC3-I ratios, is induced in the liver, heart, and spleen over 24 h after CLP. In the liver, autophagosome formation peaks at 6 h and declines by 24 h. Immunofluorescent localization of GFP-LC3 dots (alone and with lysosome-associated membrane protein type 1 (LAMP1)), as well as electron microscopic examination, demonstrate that both autophagosomes and autolysosomes are increased after CLP, suggesting that intact autophagy mechanisms operate in the liver in this model. Furthermore, inhibition of autophagy process by chloroquine administration immediately after CLP resulted in elevated serum transaminase levels and a significant increase in mortality.ConclusionsAll autophagy-related processes are properly activated in the liver in a mouse model of sepsis; autophagy appears to play a protective role in septic animals.

Tumor-suppressive microRNA-29a inhibits cancer cell migration and invasion via targeting HSP47 in cervical squamous cell carcinoma

Our recent studies of microRNA (miRNA) expression signatures indicated that microRNA-29a (miR-29a) was significantly downregulated in several types of human cancers, suggesting that miR-29a may be a putative tumor-suppressive miRNA in human cancers. The aim of this study was to investigate the functional significance of miR-29a in cervical squamous cell carcinoma (SCC) and to identify novel miR-29a-regulated cancer pathways and target genes involved in cervical SCC oncogenesis and metastasis. Restoration of miR-29a in cervical cancer cell lines (CaSKi, HeLa, ME180 and Yumoto) revealed that this miRNA significantly inhibited cancer cell migration and invasion. Gene expression data and in silico analysis demonstrated that heat-shock protein 47 (HSP47), a member of the serpin superfamily of serine proteinase inhibitors and a molecular chaperone involved in the maturation of collagen molecules, was a potential target of miR-29a regulation. Luciferase reporter assays showed that miR-29a directly regulated HSP47. Moreover, silencing of the HSP47 gene significantly inhibited cell migration and invasion in cancer cells and the expression of HSP47 was upregulated in cancer tissues and cervical intraepithelial neoplasia (CIN), as demonstrated by immunostaining. Downregulation of miR-29a was a frequent event in cervical SCC and miR-29a acted as a tumor suppressor by directly targeting HSP47. Recognition of tumor-suppressive miRNA-regulated molecular targets provides new insights into the potential mechanisms of cervical SCC oncogenesis and metastasis and suggests novel therapeutic strategies for treatment of this disease.

Tumor suppressive microRNA-133a regulates novel targets: Moesin contributes to cancer cell proliferation and invasion in head and neck squamous cell carcinoma

Recently, many studies suggest that microRNAs (miRNAs) contribute to the development, invasion and metastasis of various types of human cancers. Our recent study revealed that expression of microRNA-133a (miR-133a) was significantly reduced in head and neck squamous cell carcinoma (HNSCC) and that restoration of miR-133a inhibited cell proliferation, migration and invasion in HNSCC cell lines, suggesting that miR-133a function as a tumor suppressor. Genome-wide gene expression analysis of miR-133a transfectants and TargetScan database showed that moesin (MSN) was a promising candidate of miR-133a target gene. MSN is a member of the ERM (ezrin, radixin and moesin) protein family and ERM function as cross-linkers between plasma membrane and actin-based cytoskeleton. The functions of MSN in cancers are controversial in previous reports. In this study, we focused on MSN and investigated whether MSN was regulated by tumor suppressive miR-133a and contributed to HNSCC oncogenesis. Restoration of miR-133a in HNSCC cell lines (FaDu, HSC3, IMC-3 and SAS) suppressed the MSN expression both in mRNA and protein level. Silencing study of MSN in HNSCC cell lines demonstrated significant inhibitions of cell proliferation, migration and invasion activities in si-MSN transfectants. In clinical specimen with HNSCC, the expression level of MSN was significantly up-regulated in cancer tissues compared to adjacent non-cancerous tissues. These data suggest that MSN may function as oncogene and is regulated by tumor suppressive miR-133a. Our analysis data of novel tumor-suppressive miR-133a-mediated cancer pathways could provide new insights into the potential mechanisms of HNSCC oncogenesis.

Actin-related protein 2/3 complex subunit 5 (ARPC5) contributes to cell migration and invasion and is directly regulated by tumor-suppressive microRNA-133a in head and neck squamous cell carcinoma

Our expression signatures of human cancers including head and neck squamous cell carcinoma (HNSCC) demonstrated that downregulation of microRNA-133a (miR-133a) were frequently observed in cancer cells. The restoration of miR-133a in cancer cells revealed that it functions as a tumor suppressor. In this study, we investigated the novel molecular targets of miR-133a in HNSCC cancer cells and its oncogenic function, especially as it contributes to cancer cell migration and invasion. The genome-wide gene expression analysis and bioinformatics study showed that actin-related protein 2/3 complex subunit 5 (ARPC5) is a candidate target of miR-133a. Furthermore, luciferase reporter assay demonstrated that ARPC5 is directly regulated by miR-133a. Silencing of ARPC5 revealed significant inhibition of cell migration and invasion in HNSCC cell lines, SAS, HSC3 and IMC-3. In HSC3 cells, restoration of miR-133a or silencing ARPC5 led to a reorganization of the actin cytoskeleton and a subsequent change in cell morphology to a round, bleb-like shape. The expression levels of ARPC5 were significantly higher in HNSCC tissues than in non-cancer tissues. Immunohistochemistry showed that the levels of ARPC5 expression were significantly higher in invasive cancer cells. ARPC5 contributed to cancer cell migration and invasion in HNSCC and this gene was directly regulated by miR-133a. Our analysis of novel tumor-suppressive miR‑133a-mediated cancer pathways provides new insights into the potential mechanisms of HNSCC oncogenesis.

Prickle promotes neurite outgrowth via the Dishevelled dependent pathway in C1300 cells

Murine Prickle1 and Prickle2 belong to the planar cell polarity genes. Prickle2 but not Prickle1 gene expression was induced in C1300 neuroblastoma cells during neurite-like process formation induced by retinoic acid (RA). Over-expression of Prickle1 or Prickle2 in C1300 cells induced striking neurite-like process formation in the absence of RA. Since Prickle binds to Dishevelled (Dvl) to induce its degradation in Drosophila, we examined the participation of Dvl protein in the neurite-like process formation of C1300 cells. Upon induction of the neurite-like process formation, the amount of Dvl1 protein decreased. Prickle1 and Prickle2 could associate with Dvl1 and over-expression of Prickle1 or Prickle2 resulted in the reduction of Dvl1 protein in C1300 cells. Furthermore, over-expression of Dvl1 in C1300 cells prevented the neurite-like process formation induced by Prickle1 or Prickle2 over-expression. Thus, Prickle1 and Prickle2 promote neurite-like process formation of C1300 cells via the Dvl1 dependent mechanism.