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miRNAs are noncoding RNAs with abnormal expression in breast cancer; their expression in high-risk benign breast tissue may relate to breast cancer risk. We examined miRNA profiles in contralateral unaffected breasts (CUB) of patients with breast cancer and validated resulting candidates in two additional sample sets. Expression profiles of 754 mature miRNAs were examined using TaqMan Low Density Arrays in 30 breast cancer samples [15 estrogen receptor (ER)-positive and 15 ER-negative] and paired CUBs and 15 reduction mammoplasty controls. Pairwise comparisons identified miRNAs with significantly differential expression. Seven candidate miRNAs were examined using qRT-PCR in a second CUB sample set (40 cases, 20 ER+, 20 ER-) and 20 reduction mammoplasty controls. Further validation was performed in 80 benign breast biopsy (BBB) samples; 40 from cases who subsequently developed breast cancer and 40 from controls who did not. Logistic regression, using tertiles of miRNA expression, was used to discriminate cases from controls. Seven miRNAs were differentially expressed in tumors and CUBs versus reduction mammoplasty samples. Among them, miR-18a and miR-210 were validated in the second CUB set, showing significantly higher expression in tumor and CUBs than in reduction mammoplasty controls. The expression of miR-18a and miR-210 was also significantly higher in BBB cases than in BBB controls. When both miR-18a and miR-210 were expressed in the upper tertiles in BBB, OR for subsequent cancer was 3.20, P = 0.023. miR-18a and miR-210 are expressed at higher levels in CUBs of patients with breast cancer, and in BBB prior to cancer development, and are therefore candidate breast cancer risk biomarkers. Cancer Prev Res; 10(1); 89-97. ©2016 AACR.
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Neoplasias de la Mama/patología , Carcinogénesis/patología , MicroARNs/genética , Adulto , Anciano , Biomarcadores de Tumor/metabolismo , Biopsia , Mama , Progresión de la Enfermedad , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/metabolismo , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores de Estrógenos/metabolismoRESUMEN
MG-RAST (http://metagenomics.anl.gov) is an open-submission data portal for processing, analyzing, sharing and disseminating metagenomic datasets. The system currently hosts over 200,000 datasets and is continuously updated. The volume of submissions has increased 4-fold over the past 24 months, now averaging 4 terabasepairs per month. In addition to several new features, we report changes to the analysis workflow and the technologies used to scale the pipeline up to the required throughput levels. To show possible uses for the data from MG-RAST, we present several examples integrating data and analyses from MG-RAST into popular third-party analysis tools or sequence alignment tools.
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Bases de Datos de Ácidos Nucleicos , Metagenómica , Internet , Alineación de SecuenciaRESUMEN
Metagenomic sequencing has produced significant amounts of data in recent years. For example, as of summer 2013, MG-RAST has been used to annotate over 110,000 data sets totaling over 43 Terabases. With metagenomic sequencing finding even wider adoption in the scientific community, the existing web-based analysis tools and infrastructure in MG-RAST provide limited capability for data retrieval and analysis, such as comparative analysis between multiple data sets. Moreover, although the system provides many analysis tools, it is not comprehensive. By opening MG-RAST up via a web services API (application programmers interface) we have greatly expanded access to MG-RAST data, as well as provided a mechanism for the use of third-party analysis tools with MG-RAST data. This RESTful API makes all data and data objects created by the MG-RAST pipeline accessible as JSON objects. As part of the DOE Systems Biology Knowledgebase project (KBase, http://kbase.us) we have implemented a web services API for MG-RAST. This API complements the existing MG-RAST web interface and constitutes the basis of KBase's microbial community capabilities. In addition, the API exposes a comprehensive collection of data to programmers. This API, which uses a RESTful (Representational State Transfer) implementation, is compatible with most programming environments and should be easy to use for end users and third parties. It provides comprehensive access to sequence data, quality control results, annotations, and many other data types. Where feasible, we have used standards to expose data and metadata. Code examples are provided in a number of languages both to show the versatility of the API and to provide a starting point for users. We present an API that exposes the data in MG-RAST for consumption by our users, greatly enhancing the utility of the MG-RAST service.
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Sistemas de Administración de Bases de Datos , Bases de Datos Genéticas , Genoma Bacteriano/genética , Metagenómica/métodos , Interfaz Usuario-Computador , Internet , Anotación de Secuencia Molecular/métodos , Programas InformáticosRESUMEN
BACKGROUND: As the impact and prevalence of large-scale metagenomic surveys grow, so does the acute need for more complete and standards compliant metadata. Metadata (data describing data) provides an essential complement to experimental data, helping to answer questions about its source, mode of collection, and reliability. Metadata collection and interpretation have become vital to the genomics and metagenomics communities, but considerable challenges remain, including exchange, curation, and distribution. Currently, tools are available for capturing basic field metadata during sampling, and for storing, updating and viewing it. Unfortunately, these tools are not specifically designed for metagenomic surveys; in particular, they lack the appropriate metadata collection templates, a centralized storage repository, and a unique ID linking system that can be used to easily port complete and compatible metagenomic metadata into widely used assembly and sequence analysis tools. RESULTS: Metazen was developed as a comprehensive framework designed to enable metadata capture for metagenomic sequencing projects. Specifically, Metazen provides a rapid, easy-to-use portal to encourage early deposition of project and sample metadata. CONCLUSIONS: Metazen is an interactive tool that aids users in recording their metadata in a complete and valid format. A defined set of mandatory fields captures vital information, while the option to add fields provides flexibility.
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The epithelium lining the epididymis has a pivotal role in ensuring a luminal environment that can support normal sperm maturation. Many of the individual genes that encode proteins involved in establishing the epididymal luminal fluid are well characterized. They include ion channels, ion exchangers, transporters, and solute carriers. However, the molecular mechanisms that coordinate expression of these genes and modulate their activities in response to biological stimuli are less well understood. To identify cis-regulatory elements for genes expressed in human epididymis epithelial cells, we generated genome-wide maps of open chromatin by DNase-seq. This analysis identified 33,542 epididymis-selective DNase I hypersensitive sites (DHS), which were not evident in five cell types of different lineages. Identification of genes with epididymis-selective DHS at their promoters revealed gene pathways that are active in immature epididymis epithelial cells. These include processes correlating with epithelial function and also others with specific roles in the epididymis, including retinol metabolism and ascorbate and aldarate metabolism. Peaks of epididymis-selective chromatin were seen in the androgen receptor gene and the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which has a critical role in regulating ion transport across the epididymis epithelium. In silico prediction of transcription factor binding sites that were overrepresented in epididymis-selective DHS identified epithelial transcription factors, including ELF5 and ELF3, the androgen receptor, Pax2, and Sox9, as components of epididymis transcriptional networks. Active genes, which are targets of each transcription factor, reveal important biological processes in the epididymis epithelium.
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Ensamble y Desensamble de Cromatina , Epidídimo/metabolismo , Células Epiteliales/metabolismo , Regulación del Desarrollo de la Expresión Génica , Infertilidad Masculina/genética , Espermatogénesis , Células Cultivadas , Mapeo Cromosómico , Biología Computacional , ADN Intergénico , Epidídimo/citología , Epidídimo/crecimiento & desarrollo , Epidídimo/fisiopatología , Células Epiteliales/citología , Sistemas Especialistas , Feto/citología , Perfilación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Humanos , Infertilidad Masculina/metabolismo , Infertilidad Masculina/fisiopatología , Masculino , Nucleosomas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Especificidad de Órganos , Regiones Promotoras GenéticasRESUMEN
The democratized world of sequencing is leading to numerous data analysis challenges; MG-RAST addresses many of these challenges for diverse datasets, including amplicon datasets, shotgun metagenomes, and metatranscriptomes. The changes from version 2 to version 3 include the addition of a dedicated gene calling stage using FragGenescan, clustering of predicted proteins at 90% identity, and the use of BLAT for the computation of similarities. Together with changes in the underlying software infrastructure, this has enabled the dramatic scaling up of pipeline throughput while remaining on a limited hardware budget. The Web-based service allows upload, fully automated analysis, and visualization of results. As a result of the plummeting cost of sequencing and the readily available analytical power of MG-RAST, over 78,000 metagenomic datasets have been analyzed, with over 12,000 of them publicly available in MG-RAST.
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Biología Computacional/métodos , Metagenómica , Programas Informáticos , Bacterias/clasificación , Bacterias/genética , Genoma Bacteriano , Secuenciación de Nucleótidos de Alto Rendimiento , InternetRESUMEN
Nucleosome positioning on the chromatin strand plays a critical role in regulating accessibility of DNA to transcription factors and chromatin modifying enzymes. Hence, detailed information on nucleosome depletion or movement at cis-acting regulatory elements has the potential to identify predicted binding sites for trans-acting factors. Using a novel method based on enrichment of mononucleosomal DNA by bacterial artificial chromosome hybridization, we mapped nucleosome positions by deep sequencing across 250 kb, encompassing the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR shows tight tissue-specific regulation of expression, which is largely determined by cis-regulatory elements that lie outside the gene promoter. Although multiple elements are known, the repertoire of transcription factors that interact with these sites to activate or repress CFTR expression remains incomplete. Here, we show that specific nucleosome depletion corresponds to well-characterized binding sites for known trans-acting factors, including hepatocyte nuclear factor 1, Forkhead box A1 and CCCTC-binding factor. Moreover, the cell-type selective nucleosome positioning is effective in predicting binding sites for novel interacting factors, such as BAF155. Finally, we identify transcription factor binding sites that are overrepresented in regions where nucleosomes are depleted in a cell-specific manner. This approach recognizes the glucocorticoid receptor as a novel trans-acting factor that regulates CFTR expression in vivo.
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Mapeo Cromosómico , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Silenciador del Gen , Nucleosomas/metabolismo , Receptores de Glucocorticoides/fisiología , Sitios de Unión , Factor de Unión a CCCTC , Células CACO-2 , Inmunoprecipitación de Cromatina , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Dexametasona/farmacología , Sitios Genéticos , Glucocorticoides/farmacología , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Humanos , Nucleosomas/genética , Unión Proteica , Receptores de Glucocorticoides/metabolismo , Proteínas Represoras/metabolismo , Elementos de Respuesta , Análisis de Secuencia de ADN , Factores de Transcripción/metabolismoRESUMEN
Access to regulatory elements of the genome can be inhibited by nucleosome core particles arranged along the DNA strand. Hence, sites that are accessible by transcription factors may be located by using nuclease digestion to identify the relative nucleosome occupancy of a genomic region. In order to define novel cis regulatory elements in the â¼2.7-kb promoter region of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, we define its nucleosome occupancy. This profile reveals the precise positions of nucleosome-free regions (NFRs), both cell-type specific and others apparently unrelated to CFTR-expression level and offer the first high-resolution map of the chromatin structure of the entire CFTR promoter in relevant cell types. Several of these NFRs are strongly bound by nuclear factors in a sequence-specific manner, and directly influence CFTR promoter activity. Sequences within the NFR1 and NFR4 elements are highly conserved in many human gene promoters. Moreover, NFR1 contributes to promoter activity of another gene, angiopoietin-like 3 (ANGPTL3), while NFR4 is constitutively nucleosome-free in promoters genome wide. Conserved motifs within NFRs of the CFTR promoter also show a high level of protection from DNase I digestion genome-wide, and likely have important roles in the positioning of nucleosome core particles more generally.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Nucleosomas/metabolismo , Regiones Promotoras Genéticas , Secuencia de Bases , Células Cultivadas , Secuencia Conservada , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Humanos , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
BACKGROUND: Distal cell-type-specific regulatory elements may be located at very large distances from the genes that they control and are often hidden within intergenic regions or in introns of other genes. The development of methods that enable mapping of regions of open chromatin genome wide has greatly advanced the identification and characterisation of these elements. METHODS: Here we use DNase I hypersensitivity mapping followed by deep sequencing (DNase-seq) to generate a map of open chromatin in primary human tracheal epithelial (HTE) cells and use bioinformatic approaches to characterise the distribution of these sites within the genome and with respect to gene promoters, intronic and intergenic regions. RESULTS: Genes with HTE-selective open chromatin at their promoters were associated with multiple pathways of epithelial function and differentiation. The data predict novel cell-type-specific regulatory elements for genes involved in HTE cell function, such as structural proteins and ion channels, and the transcription factors that may interact with them to control gene expression. Moreover, the map of open chromatin can identify the location of potentially critical regulatory elements in genome-wide association studies (GWAS) in which the strongest association is with single nucleotide polymorphisms in non-coding regions of the genome. We demonstrate its relevance to a recent GWAS that identifies modifiers of cystic fibrosis lung disease severity. CONCLUSION: Since HTE cells have many functional similarities with bronchial epithelial cells and other differentiated cells in the respiratory epithelium, these data are of direct relevance to elucidating the molecular basis of normal lung function and lung disease.
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Cromatina/genética , Células Epiteliales/metabolismo , Regulación de la Expresión Génica , Pulmón/fisiología , Mucosa Respiratoria/metabolismo , Tráquea/citología , Mapeo Cromosómico/métodos , Biología Computacional , Desoxirribonucleasa I , Estudio de Asociación del Genoma Completo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Pulmón/metabolismoRESUMEN
INTRODUCTION: We have examined expression of microRNAs (miRNAs) in ependymomas to identify molecular markers of value for clinical management. miRNAs are non-coding RNAs that can block mRNA translation and affect mRNA stability. Changes in the expression of miRNAs have been correlated with many human cancers. MATERIALS AND METHODS: We have utilized TaqMan Low Density Arrays to evaluate the expression of 365 miRNAs in ependymomas and normal brain tissue. We first demonstrated the similarity of expression profiles of paired frozen tissue (FT) and paraffin-embedded specimens (FFPE). We compared the miRNA expression profiles of 34 FFPE ependymoma samples with 8 microdissected normal brain tissue specimens enriched for ependymal cells. miRNA expression profiles were then correlated with tumor location, histology and other clinicopathological features. RESULTS: We have identified miRNAs that are over-expressed in ependymomas, such as miR-135a and miR-17-5p, and down-regulated, such as miR-383 and miR-485-5p. We have also uncovered associations between expression of specific miRNAs which portend a worse prognosis. For example, we have identified a cluster of miRNAs on human chromosome 14q32 that is associated with time to relapse. We also found that miR-203 is an independent marker for relapse compared to the parameters that are currently used. Additionally, we have identified three miRNAs (let-7d, miR-596 and miR-367) that strongly correlate to overall survival. CONCLUSION: We have identified miRNAs that are differentially expressed in ependymomas compared with normal ependymal tissue. We have also uncovered significant associations of miRNAs with clinical behavior. This is the first report of clinically relevant miRNAs in ependymomas.
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Biomarcadores de Tumor/genética , Ependimoma/genética , MicroARNs/genética , Adolescente , Biomarcadores de Tumor/metabolismo , Estudios de Casos y Controles , Niño , Preescolar , Biología Computacional , Ependimoma/patología , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Lactante , Masculino , MicroARNs/metabolismo , Análisis Multivariante , Pronóstico , Modelos de Riesgos Proporcionales , Análisis de Regresión , Análisis de SupervivenciaRESUMEN
A limited number of reports have investigated the role of microRNAs in osteosarcoma. In this study, we performed miRNA expression profiling of osteosarcoma cell lines, tumor samples, and normal human osteoblasts. Twenty-two differentially expressed microRNAs were identified using high throughput real-time PCR analysis, and 4 (miR-135b, miR-150, miR-542-5p, and miR-652) were confirmed and validated in a different group of tumors. Both miR-135b and miR-150 have been previously shown to be important in cancer. We hypothesize that dysregulation of differentially expressed microRNAs may contribute to tumorigenesis. They might also represent molecular biomarkers or targets for drug development in osteosarcoma.
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Melanoma progression requires deregulation of gene expression by currently uncharacterized epigenetic mechanisms. A mouse model based on changes in cell microenvironment was developed by our group to study melanocyte malignant transformation. Melanoma cell lines (4C11- and 4C11+) were obtained as result of 5 sequential anchorage blockades of non-tumorigenic melan-a melanocytes. Melan-a cells submitted to 4 de-adhesion cycles were also established (4C), are non-tumorigenic and represent an intermediary phase of tumor progression. The aim of this work was to identify factors contributing to epigenetic modifications in early and later phases of malignant transformation induced by anchorage impediment. Epigenetic alterations occur early in tumorigenesis; 4C cell line shows changes in global and gene-specific DNA methylation and histone marks. Many histone modifications differ between melan-a, 4C, 4C11- (non-metastatic melanoma cell line) and 4C11+ (metastatic melanoma cell line) which could be associated with changes in gene and microRNA expression. These epigenetic alterations seem to play a key role in malignant transformation since melanocytes treated with 5-Aza-2'-deoxycytidine before each anchorage blockade do not transform. Some epigenetic changes seem to be also responsible for the maintenance of malignant phenotype, since melanoma cell lines (4C11- and 4C11+) treated in vitro with 5-Aza-2'-deoxycytidine or Trichostatin A showed reduction of tumor growth in vivo. Changes in gene expression reflecting cell adaptation to new environment were also observed. We propose a model in which sustained microenvironmental stress in melanocytes results in epigenetic reprogramming. Thus, after adaptation, cells may acquire epigenetic marks that could contribute to the establishment of a malignant phenotype.
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Transformación Celular Neoplásica/genética , Metilación de ADN , Epigénesis Genética , Histonas/metabolismo , Melanocitos/fisiología , Animales , Línea Celular , Transformación Celular Neoplásica/inducido químicamente , Regulación Neoplásica de la Expresión Génica , RatonesRESUMEN
The mechanism(s) behind folate rescue of neural tube closure are not well understood. In this study we show that maternal intake of folate prior to conception reverses the proliferation potential of neural crest stem cells in homozygous Splotch embryos (Sp(-/-)) via epigenetic mechanisms. It is also shown that the pattern of differentiation seen in these cells is similar to wild-type (WT). Cells from open caudal neural tubes of Sp(-/-) embryos exhibit increased H3K27 methylation and decreased expression of KDM6B possibly due to up-regulation of KDM6B targeting micro-RNAs such as miR-138, miR-148a, miR-185, and miR-339-5p. In our model, folate reversed these epigenetic marks in folate-rescued Sp(-/-) embryos. Using tissue from caudal neural tubes of murine embryos we also examined H3K27me2 and KDM6B association with Hes1 and Neurog2 promoters at embryonic day E10.5, the proliferative stage, and E12.5, when neural differentiation begins. In Sp(-/-) embryos compared with WT, levels of H3K27me2 associated with the Hes1 promoter were increased at E10.5, and levels associated with the Neurog2 promoter were increased at E12.5. KDM6B association with Hes1 and Neurog2 promoters was inversely related to H3K27me2 levels. These epigenetic changes were reversed in folate-rescued Sp(-/-) embryos. Thus, one of the mechanisms by which folate may rescue the Sp(-/-) phenotype is by increasing the expression of KDM6B, which in turn decreases H3K27 methylation marks on Hes1 and Neurog2 promoters thereby affecting gene transcription.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Ácido Fólico/administración & dosificación , Proteínas de Homeodominio/genética , Proteínas del Tejido Nervioso/genética , Tubo Neural/efectos de los fármacos , Tubo Neural/embriología , Regiones Promotoras Genéticas/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Western Blotting , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Sistema Nervioso Central/embriología , Ensamble y Desensamble de Cromatina/genética , Inmunoprecipitación de Cromatina , Embrión de Mamíferos/citología , Embrión de Mamíferos/efectos de los fármacos , Embrión de Mamíferos/metabolismo , Epigenómica , Femenino , Técnica del Anticuerpo Fluorescente , Ácido Fólico/farmacología , Histonas/genética , Histonas/metabolismo , Proteínas de Homeodominio/metabolismo , Técnicas para Inmunoenzimas , Inmunoprecipitación , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Luciferasas/metabolismo , Masculino , Metilación , Ratones , Ratones Endogámicos C57BL , MicroARNs/fisiología , Proteínas del Tejido Nervioso/metabolismo , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Defectos del Tubo Neural/prevención & control , Factor de Transcripción PAX3 , Factores de Transcripción Paired Box/fisiología , Regiones Promotoras Genéticas/efectos de los fármacos , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción HES-1 , Complejo Vitamínico B/administración & dosificación , Complejo Vitamínico B/farmacologíaRESUMEN
BACKGROUND: Chondrosarcomas are malignant cartilage tumors that do not respond to traditional chemotherapy or radiation. The 5-year survival rate of histologic grade III chondrosarcoma is less than 30%. An animal model of chondrosarcoma has been established--namely, the Swarm Rat Chondrosarcoma (SRC)--and shown to resemble the human disease. Previous studies with this model revealed that tumor microenvironment could significantly influence chondrosarcoma malignancy. METHODS: To examine the effect of the microenvironment, SRC tumors were initiated at different transplantation sites. Pyrosequencing assays were utilized to assess the DNA methylation of the tumors, and SAGE libraries were constructed and sequenced to determine the gene expression profiles of the tumors. Based on the gene expression analysis, subsequent functional assays were designed to determine the relevancy of the specific genes in the development and progression of the SRC. RESULTS: The site of transplantation had a significant impact on the epigenetic and gene expression profiles of SRC tumors. Our analyses revealed that SRC tumors were hypomethylated compared to control tissue, and that tumors at each transplantation site had a unique expression profile. Subsequent functional analysis of differentially expressed genes, albeit preliminary, provided some insight into the role that thymosin-ß4, c-fos, and CTGF may play in chondrosarcoma development and progression. CONCLUSION: This report describes the first global molecular characterization of the SRC model, and it demonstrates that the tumor microenvironment can induce epigenetic alterations and changes in gene expression in the SRC tumors. We documented changes in gene expression that accompany changes in tumor phenotype, and these gene expression changes provide insight into the pathways that may play a role in the development and progression of chondrosarcoma. Furthermore, specific functional analysis indicates that thymosin-ß4 may have a role in chondrosarcoma metastasis.
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Biomarcadores de Tumor/genética , Condrosarcoma/genética , Epigénesis Genética , Perfilación de la Expresión Génica , Neoplasias Pulmonares/etiología , Tibia/patología , Animales , Biomarcadores de Tumor/metabolismo , Western Blotting , Cartílago/metabolismo , Cartílago/patología , Condrosarcoma/metabolismo , Condrosarcoma/patología , Factor de Crecimiento del Tejido Conjuntivo/genética , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Metilación de ADN , Genes fos/fisiología , Humanos , Inyecciones Subcutáneas , Neoplasias Pulmonares/secundario , Masculino , Ratones , Ratones Desnudos , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Ratas , Ratas Sprague-Dawley , Timosina/genética , Timosina/metabolismo , Tibia/metabolismo , Células Tumorales Cultivadas/trasplanteRESUMEN
PURPOSE: To understand the changes in gene expression in polycythemia vera (PV) progenitor cells and their relationship to JAK2V617F. EXPERIMENTAL DESIGN: Messenger RNA isolated from CD34(+) cells from nine PV patients and normal controls was profiled using Affymetrix arrays. Gene expression change mediated by JAK2V617F was determined by profiling CD34(+) cells transduced with the kinase and by analysis of leukemia cell lines harboring JAK2V617F, treated with an inhibitor. RESULTS: A PV expression signature was enriched for genes involved in hematopoietic development, inflammatory responses, and cell proliferation. By quantitative reverse transcription-PCR, 23 genes were consistently deregulated in all patient samples. Several of these genes such as WT1 and KLF4 were regulated by JAK2, whereas others such as NFIB and EVI1 seemed to be deregulated in PV by a JAK2-independent mechanism. Using cell line models and comparing gene expression profiles of cell lines and PV CD34(+) PV specimens, we have identified panels of 14 JAK2-dependent genes and 12 JAK2-independent genes. These two 14- and 12-gene sets could separate not only PV from normal CD34(+) specimens, but also other MPN such as essential thrombocytosis and primary myelofibrosis from their normal counterparts. CONCLUSIONS: A subset of the aberrant gene expression in PV progenitor cells can be attributed to the action of the mutant kinase, but there remain a significant number of genes characteristic of the disease but deregulated by as yet unknown mechanisms. Genes deregulated in PV as a result of the action of JAK2V617F or independent of the kinase may represent other targets for therapy.
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Perfilación de la Expresión Génica , Janus Quinasa 2/genética , Mutación , Policitemia Vera/genética , Sustitución de Aminoácidos , Antígenos CD34/genética , Antígenos CD34/metabolismo , Células de la Médula Ósea/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Análisis por Conglomerados , Células Eritroides/citología , Células Eritroides/metabolismo , Eritropoyetina/farmacología , Humanos , Janus Quinasa 2/metabolismo , Factor 4 Similar a Kruppel , Análisis de Secuencia por Matrices de Oligonucleótidos , Policitemia Vera/sangre , Policitemia Vera/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
PURPOSE: The aim of this study is to search for new therapeutic targets for atypical teratoid-rhabdoid tumors (ATRT). METHODS: To achieve this, we compared the expression of 365 microRNAs among ATRT, medulloblastomas, and normal brain. RESULTS: MiR-221 and miR-222 were within the top differentially expressed microRNAs. The deregulated expression of miR221/222 was demonstrated to inhibit the expression of the tumor suppressor and inhibitor of cell cycle p27(Kip1). Here, we demonstrated the negative regulation of p27(Kip1) by miR-221/222 in ATRT using microarray, real-time reverse transcriptase polymerase chain reaction, and immunohistochemistry. CONCLUSION: As anti-miR therapy was recently proposed as an alternative treatment for cancer, these findings suggest that anti-miR-221/222 therapy might have therapeutic potential in ATRT.
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Neoplasias Encefálicas/metabolismo , Encéfalo/metabolismo , MicroARNs/metabolismo , Tumor Rabdoide/metabolismo , Teratoma/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Proteínas Cromosómicas no Histona/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas de Unión al ADN/metabolismo , Femenino , Humanos , Inmunohistoquímica , Lactante , Recién Nacido , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tumor Rabdoide/tratamiento farmacológico , Proteína SMARCB1 , Teratoma/tratamiento farmacológico , Factores de Transcripción/metabolismoRESUMEN
The cystic fibrosis transmembrane conductance regulator (CFTR) gene is driven by a promoter that cannot alone account for the temporal and tissue-specific regulation of the gene. This has led to the search for additional regulatory elements that cooperate with the basal promoter to achieve coordinated expression. We previously identified two alternative upstream exons of the gene that were mutually exclusive of the first exon, and one of which showed temporal regulation in the human and sheep lung. We now demonstrate that this alternative splice product generates a stable protein, which initiates translation at an ATG in exon 4, and thus lacks the N terminus of CFTR. The other splice variant inhibits translation of the protein. In a search for the promoter used by the upstream exons, we identified a novel element that contributes to the activity of the basal CFTR promoter in airway epithelial cells, but does not function independently. Finally, we demonstrate that, in primary airway cells, skin fibroblasts, and both airway and intestinal cell lines, the CFTR promoter is unmethylated, irrespective of CFTR expression status. Thus, methylation is not the main cause of inactivation of CFTR transcription.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/genética , Metilación de ADN , Regulación de la Expresión Génica , Regiones Promotoras Genéticas/genética , Transcripción Genética/genética , Empalme Alternativo , Secuencia de Bases , Bronquios/citología , Bronquios/metabolismo , Células CACO-2/metabolismo , Células Cultivadas , Fibrosis Quística/patología , Células Epiteliales/metabolismo , Exones/genética , Humanos , Intestino Delgado/metabolismo , Luciferasas/metabolismo , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Secuencias Reguladoras de Ácidos NucleicosRESUMEN
Abnormal patterns of DNA methylation are observed in several types of human cancer. While localized DNA methylation of CpG islands has been associated with gene silencing, the effect that genome-wide loss of methylation has on tumorigenesis is not completely known. To examine its effect on tumorigenesis, we induced DNA demethylation in a rat model of human chondrosarcoma using 5-aza-2-deoxycytidine. Rat specific pyrosequencing assays were utilized to assess the methylation levels in both LINEs and satellite DNA sequences following 5-aza-2-deoxycytidine treatment. Loss of DNA methylation was accompanied by an increase in invasiveness of the rat chondrosarcoma cells, in vitro, as well as by an increase in tumor growth in vivo. Subsequent microarray analysis provided insight into the gene expression changes that result from 5-aza-2-deoxycytidine induced DNA demethylation. In particular, two genes that may function in tumorigenesis, sox-2 and midkine, were expressed at low levels in control cells but upon 5-aza-2-deoxycytidine treatment these genes became overexpressed. Promoter region DNA analysis revealed that these genes were methylated in control cells but became demethylated following 5-aza-2-deoxycytidine treatment. Following withdrawal of 5-aza-2-deoxycytidine, the rat chondrosarcoma cells reestablished global DNA methylation levels that were comparable to that of control cells. Concurrently, invasiveness of the rat chondrosarcoma cells, in vitro, decreased to a level indistinguishable to that of control cells. Taken together these experiments demonstrate that global DNA hypomethylation induced by 5-aza-2-deoxycytidine may promote specific aspects of tumorigenesis in rat chondrosarcoma cells.
Asunto(s)
Azacitidina/análogos & derivados , Condrosarcoma/genética , Condrosarcoma/patología , Metilación de ADN/efectos de los fármacos , Animales , Azacitidina/farmacología , Línea Celular Tumoral , Citocinas/genética , Citocinas/metabolismo , Decitabina , Epigénesis Genética/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Elementos de Nucleótido Esparcido Largo/genética , Ratones , Repeticiones de Microsatélite/genética , Midkina , Invasividad Neoplásica/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Análisis de Secuencia de ADN , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Base substitution occurs at a high rate at CpG dinucleotides due to the frequent methylation of CpG and the deamination of methylated cytosine to thymine. If these substitutions occur in germ cells, they constitute a heritable mutation that may eventually rise to polymorphic frequencies, hence resulting in a SNP that is methylation associated. In this study, we sought to identify clusters of methylation associated SNPs as a basis for prediction of methylation landscapes of germ cell genomes. Genomic regions enriched with methylation associated SNPs, namely "methylation associated SNP clusters", were identified with an agglomerative hierarchical clustering algorithm. Repetitive elements, segmental duplications, and syntenic tandem DNA repeats were enriched in methylation associated SNP clusters. The frequency of methylation associated SNPs in Alu Y/S elements exhibited a gradient pattern suggestive of linear spreading, being higher in proximity to methylation associated SNP clusters and lower closer to CpG islands. Interestingly, methylation associated SNP clusters were over-represented near the transcriptional initiation sites of immune response genes. We propose a de novo DNA methylation model during germ cell development whereby a pattern is established by long-range chromatic interactions through syntenic repeats combined with regional methylation spreading from methylation associated SNP clusters.
Asunto(s)
Metilación de ADN , Células Germinativas/metabolismo , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADN/métodos , Algoritmos , Islas de CpG , Humanos , Sitio de Iniciación de la TranscripciónRESUMEN
UNLABELLED: We have previously shown that the microenvironment of human embryonic stem cells (hESCs) is able to change and reprogram aggressive cancer cells to a less aggressive state. Some mechanisms implicated in the phenotypic changes observed after this exposure are mainly associated with the Nodal signaling pathway, which plays a key role in tumor cell plasticity. However, several other molecular mechanisms might be related directly and/or indirectly to these changes, including microRNA (miRNA) regulation and DNA methylation. AIM: To further explore the epigenetic mechanisms potentially underlying the phenotypic changes that occur after exposing metastatic melanoma cells to a hESC microenvironment. MATERIALS & METHODS: A total of 365 miRNAs were screened using the TaqMan® Low Density Arrays. We also evaluated whether DNA methylation could be one of the factors regulating the expression of the inhibitor of Nodal, Lefty, in hESCs (where it is highly expressed) vs melanoma cells (where it is not expressed). RESULTS: Using these experimental approaches, we identified miRNAs that are up- and down-regulated in melanoma cells exposed to a hESC microenvironment, such as miR-302a and miR-27b, respectively. We also demonstrate that Notch4 is one of the targets of miR-302a, which is upstream of Nodal. Additionally, one of the mechanisms that might explain the absence of the inhibitor of Nodal, Lefty, in cancer cells is silencing by DNA methylation, which provides new insights into the unregulated expression of Nodal in melanoma. CONCLUSION: These findings suggest that epigenetic changes such as DNA methylation and regulation by microRNAs might play a significant role in tumor cell plasticity and the metastatic phenotype.