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1.
J Pathol ; 245(4): 387-398, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29570800

RESUMO

Deregulated DNA methylation leading to transcriptional inactivation of certain genes occurs frequently in non-small-cell lung cancers (NSCLCs). As well as protein-coding genes, microRNA (miRNA)-coding genes may be targets for methylation in NSCLCs; however, the number of known methylated miRNA genes is still small. Thus, we investigated methylation of miRNA genes in primary tumour (TU) samples and corresponding non-malignant lung tissue (NL) samples of 50 NSCLC patients by using methylated DNA immunoprecipitation followed by custom-designed tiling microarray analyses (MeDIP-chip), and 252 differentially methylated probes between TU samples and NL samples were identified. These probes were annotated, which resulted in the identification of 34 miRNA genes with increased methylation in TU samples. Some of these miRNA genes were already known to be methylated in NSCLCs (e.g. those encoding miR-9-3 and miR-124), but methylation of the vast majority of them was previously unknown. We selected six miRNA genes (those encoding miR-10b, miR-1179, miR-137, miR-572, miR-3150b, and miR-129-2) for gene-specific methylation analyses in TU samples and corresponding NL samples of 104 NSCLC patients, and observed a statistically significant increase in methylation of these genes in TU samples (p < 0.0001). In silico target prediction of the six miRNAs identified several oncogenic/cell proliferation-promoting factors (e.g. CCNE1 as an miR-1179 target). To investigate whether miR-1179 indeed targets CCNE1, we transfected miR-1179 gene mimics into CCNE1-expressing NSCLC cells, and observed downregulated CCNE1 mRNA expression in these cells as compared with control cells. Similar effects on cyclin E1 expression were seen in western blot analyses. In addition, we found a statistically significant reduction in the growth of NSCLC cells transfected with miR-1179 mimics as compared with control cells. In conclusion, we identified many methylated miRNA genes in NSCLC patients, and found that the miR-1179 gene is a potential tumour cell growth suppressor in NSCLCs. Overall, our findings emphasize the impact of miRNA gene methylation on the pathogenesis of NSCLCs. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Metilação de DNA , Neoplasias Pulmonares/genética , MicroRNAs/genética , Células A549 , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/cirurgia , Proliferação de Células/genética , Imunoprecipitação da Cromatina/métodos , Ilhas de CpG , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Predisposição Genética para Doença , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/cirurgia , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Transdução de Sinais/genética
2.
Mol Cancer ; 16(1): 1, 2017 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-28093071

RESUMO

BACKGROUND: DNA methylation regulates together with other epigenetic mechanisms the transcriptional activity of genes and is involved in the pathogenesis of malignant diseases including lung cancer. In non-small cell lung cancer (NSCLC) various tumor suppressor genes are already known to be tumor-specifically methylated. However, from the vast majority of a large number of genes which were identified to be tumor-specifically methylated, tumor-specific methylation was unknown so far. Thus, the major aim of this study was to investigate in detail the mechanism(s) responsible for transcriptional regulation of the genes SPAG6 and L1TD1 in NSCLCs. METHODS: We analysed publically available RNA-sequencing data and performed gene expression analyses by RT-PCR. DNA methylation analyses were done by methylation-sensitive high-resolution melt analyses and bisulfite genomic sequencing. We additionally investigated protein expression using immunohistochemistry. Cell culture experiments included tumor cell growth, proliferation, viability as well as colony formation assays. Moreover, we performed xenograft experiments using immunodeficient mice. RESULTS: We observed frequent downregulation of SPAG6 and L1TD1 mRNA expression in primary tumor (TU) samples compared to corresponding non-malignant lung tissue (NL) samples of NSCLC patients. We furthermore observed re-expression of both genes after treatment with epigenetically active drugs in most NSCLC cell lines with downregulated SPAG6 and L1TD1 mRNA expression. Frequent tumor-specific DNA methylation of SPAG6 and L1TD1 was detected when we analysed TU and corresponding NL samples of NSCLC patients. ROC curve analyses demonstrated that methylation of both genes is able to distinguish between TU and NL samples of these patients. Immunohistochemistry revealed a close association between SPAG6/L1TD1 methylation and downregulated protein expression of these genes. Moreover, by performing functional assays we observed reduced cell growth, proliferation and viability of pCMV6-L1TD1 transfected NSCLC cells. In addition, reduced volumes of tumors derived from pCMV6-L1TD1 compared to pCMV6-ENTRY transfected NCI-H1975 cells were seen in a xenograft tumor model. CONCLUSIONS: Overall, our results demonstrate that SPAG6 and L1TD1 are tumor-specifically methylated in NSCLCs and that DNA methylation is involved in the transcriptional regulation of these genes. Moreover, in vitro as well as in vivo experiments revealed tumor-cell growth suppressing properties of L1TD1 in NSCLC cells.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Proteínas dos Microtúbulos/genética , Proteínas/genética , Transcrição Gênica , Animais , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/genética , Bases de Dados Genéticas , Modelos Animais de Doenças , Inativação Gênica , Xenoenxertos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Proteínas dos Microtúbulos/metabolismo , Polimorfismo de Nucleotídeo Único , Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Deleção de Sequência , Carga Tumoral , Ensaio Tumoral de Célula-Tronco
3.
J Pathol ; 237(2): 203-14, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26011651

RESUMO

Malignant pleural mesothelioma (MPM) is a devastating malignancy characterized by invasive growth and rapid recurrence. The identification and inhibition of molecular components leading to this migratory and invasive phenotype are thus essential. Accordingly, a genome-wide expression array analysis was performed on MPM cell lines and a set of 139 genes was identified as differentially expressed in cells with high versus low migratory activity. Reduced expression of the novel tumour suppressor integrin α7 (ITGA7) was found in highly motile cells. A significant negative correlation was observed between ITGA7 transcript levels and average displacement of cells. Forced overexpression of ITGA7 in MPM cells with low endogenous ITGA7 expression inhibited cell motility, providing direct evidence for the regulatory role of ITGA7 in MPM cell migration. MPM cells showed decreased ITGA7 expressions at both transcription and protein levels when compared to non-malignant mesothelial cells. The majority of MPM cell cultures displayed hypermethylation of the ITGA7 promoter when compared to mesothelial cultures. A statistically significant negative correlation between ITGA7 methylation and ITGA7 expression was also observed in MPM cells. While normal human pleura samples unambiguously expressed ITGA7, a varying level of expression was found in a panel of 200 human MPM samples. In multivariate analysis, ITGA7 expression was found to be an independent prognostic factor. Although there was no correlation between histological subtypes and ITGA7 expression, importantly, patients with high tumour cell ITGA7 expression had an increased median overall survival compared to the low- or no-expression groups (463 versus 278 days). In conclusion, our data suggest that ITGA7 is an epigenetically regulated tumour suppressor gene and a prognostic factor in human MPM.


Assuntos
Antígenos CD/metabolismo , Movimento Celular , Epigênese Genética , Cadeias alfa de Integrinas/metabolismo , Neoplasias Pulmonares/metabolismo , Mesotelioma/metabolismo , Neoplasias Pleurais/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Antígenos CD/genética , Linhagem Celular Tumoral , Metilação de DNA , Regulação para Baixo , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Cadeias alfa de Integrinas/genética , Estimativa de Kaplan-Meier , Laminina/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Mesotelioma/genética , Mesotelioma/mortalidade , Mesotelioma/patologia , Mesotelioma Maligno , Análise Multivariada , Invasividade Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Pleurais/genética , Neoplasias Pleurais/mortalidade , Neoplasias Pleurais/patologia , Prognóstico , Regiões Promotoras Genéticas , Modelos de Riscos Proporcionais , RNA Mensageiro/metabolismo , Fatores de Risco , Transdução de Sinais , Fatores de Tempo , Transfecção , Proteínas Supressoras de Tumor/genética
4.
Carcinogenesis ; 34(3): 513-21, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23172663

RESUMO

DNA methylation is part of the epigenetic gene regulation complex, which is relevant for the pathogenesis of cancer. We performed a genome-wide search for methylated CpG islands in tumors and corresponding non-malignant lung tissue samples of 101 stages I-III non-small cell lung cancer (NSCLC) patients by combining methylated DNA immunoprecipitation and microarray analysis. Overall, we identified 2414 genomic positions differentially methylated between tumor and non-malignant lung tissue samples. Ninety-seven percent of them were found to be tumor-specifically methylated. Annotation of these genomic positions resulted in the identification of 477 tumor-specifically methylated genes of which many are involved in regulation of gene transcription and cell adhesion. Tumor-specific methylation was confirmed by a gene-specific approach. In the majority of tumors, methylation of certain genes was associated with loss of their protein expression determined by immunohistochemistry. Treatment of NSCLC cells with epigenetically active drugs resulted in upregulated expression of many tumor-specifically methylated genes analyzed by gene expression microarrays suggesting that about one-third of these genes are transcriptionally regulated by methylation. Moreover, comparison of methylation results with certain clinicopathological characteristics of the patients suggests that methylation of HOXA2 and HOXA10 may be of prognostic relevance in squamous cell carcinoma (SCC) patients. In conclusion, we identified a large number of tumor-specifically methylated genes in NSCLC patients. Expression of many of them is regulated by methylation. Moreover, HOXA2 and HOXA10 methylation may serve as prognostic parameters in SCC patients. Overall, our findings emphasize the impact of methylation on the pathogenesis of NSCLCs.


Assuntos
Adenocarcinoma/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma de Células Escamosas/genética , Metilação de DNA , Neoplasias Pulmonares/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidade , Sequência de Bases , Caderinas/genética , Caderinas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/mortalidade , Mapeamento Cromossômico , Ilhas de CpG , Feminino , Regulação Neoplásica da Expressão Gênica , Genes Neoplásicos , Estudo de Associação Genômica Ampla , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidade , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Curva ROC , Análise de Sequência de DNA , Transcriptoma
5.
Oncotarget ; 6(1): 394-408, 2015 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-25504438

RESUMO

In our study, we investigated the role of ZNF677 in non-small cell lung cancers (NSCLC). By comparing ZNF677 expression in primary tumor (TU) and in the majority of cases also of corresponding non-malignant lung tissue (NL) samples from > 1,000 NSCLC patients, we found tumor-specific downregulation of ZNF677 expression (adjusted p-values < 0.001). We identified methylation as main mechanism for ZNF677 downregulation in NSCLC cells and we observed tumor-specific ZNF677 methylation in NSCLC patients (p < 0.0001). In the majority of TUs, ZNF677 methylation was associated with loss of ZNF677 expression. Moreover, ZNF677 overexpression in NSCLC cells was associated with reduced cell proliferation and cell migration. ZNF677 was identified to regulate expression of many genes mainly involved in growth hormone regulation and interferon signalling. Finally, patients with ZNF677 methylated TUs had a shorter overall survival compared to patients with ZNF677 not methylated TUs (p = 0.013). Overall, our results demonstrate that ZNF677 is trancriptionally regulated by methylation in NSCLCs, suggest that ZNF677 has tumor cell growth suppressing properties in NSCLCs and that ZNF677 methylation might serve as prognostic parameter in these patients.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Metilação de DNA/genética , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias Pulmonares/genética , Dedos de Zinco/genética , Área Sob a Curva , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Curva ROC , Análise Serial de Tecidos , Transcrição Gênica , Transfecção
6.
Clin Cancer Res ; 18(6): 1619-29, 2012 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-22282464

RESUMO

PURPOSE: The major aim of this study was to investigate the role of DNA methylation (referred to as methylation) on miRNA silencing in non-small cell lung cancers (NSCLC). EXPERIMENTAL DESIGN: We conducted microarray expression analyses of 856 miRNAs in NSCLC A549 cells before and after treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (Aza-dC) and with a combination of Aza-dC and the histone deacetylase inhibitor trichostatin A. miRNA methylation was determined in 11 NSCLC cell lines and in primary tumors and corresponding nonmalignant lung tissue samples of 101 patients with stage I-III NSCLC. RESULTS: By comparing microarray data of untreated and drug-treated A549 cells, we identified 33 miRNAs whose expression was upregulated after drug treatment and which are associated with a CpG island. Thirty (91%) of these miRNAs were found to be methylated in at least 1 of 11 NSCLC cell lines analyzed. Moreover, miR-9-3 and miR-193a were found to be tumor specifically methylated in patients with NSCLC. We observed a shorter disease-free survival of patients with miR-9-3 methylated lung squamous cell carcinoma (LSCC) than patients with miR-9-3 unmethylated LSCC by multivariate analysis [HR = 3.8; 95% confidence interval (CI), 1.3-11.2, P = 0.017] and a shorter overall survival of patients with miR-9-3 methylated LSCC than patients with miR-9-3 unmethylated LSCC by univariate analysis (P = 0.013). CONCLUSIONS: Overall, our results suggest that methylation is an important mechanism for inactivation of certain miRNAs in NSCLCs and that miR-9-3 methylation may serve as a prognostic parameter in patients with LSCC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Metilação de DNA , Neoplasias Pulmonares/genética , MicroRNAs/metabolismo , Antineoplásicos/farmacologia , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Linhagem Celular Tumoral , Decitabina , Feminino , Perfilação da Expressão Gênica , Inativação Gênica , Humanos , Ácidos Hidroxâmicos/farmacologia , Pulmão/metabolismo , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Análise de Sequência com Séries de Oligonucleotídeos
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