RESUMEN
OBJECTIVE: Our objective was to characterize the expression and function of the miR-200 family of microRNAs (miRNA) in ovarian carcinogenesis. METHODS: We used qRT-PCR to examine expression of the miR-200 miRNA family and its predicted targets, the ZEB1 and ZEB2 transcriptional repressors, in primary cultures of normal cells from the surface of the ovary and in a panel of 70 ovarian cancer tissues and 15 ovarian cancer cell lines. We studied the mechanisms of regulation of miR-200 miRNAs and ZEB transcription factors in ovarian cells using 3' UTR luciferase reporters, promoter luciferase reporters and siRNAs. RESULTS: miR-200 family members are expressed at low or negligible levels in normal ovarian surface cells and substantially increase in expression in ovarian cancer, whereas expression of ZEB1 and ZEB2 shows the opposite pattern. There is reciprocal repression between miR-200 family members and ZEB transcription factors, creating a double negative regulatory feedback loop resembling that reported in other cancer cell types. In contrast to epithelial cells from other sites, expression levels of miR-200 miRNAs and ZEB1/2 in cells from the ovarian surface are more consistent with a mesenchymal cell phenotype, potentially reflecting the mesothelial origin of the ovarian surface. CONCLUSION: Analysis of ovarian cancer tissues suggests that ovarian surface cells acquire a more epithelial miR-200-ZEB1/2 phenotype as they undergo transformation, switching from a miR-200 familyLOW and ZEB1/2HIGH state to a miR-200 familyHIGH and ZEB1/2LOW phenotype. Collectively, our data support the mesothelial-to-epithelial (Meso-E-T) model for development of ovarian cancers that arise from ovarian surface cells, as has been proposed previously on the basis of studies of protein markers.
Asunto(s)
Transformación Celular Neoplásica/genética , Proteínas de Homeodominio/biosíntesis , MicroARNs/biosíntesis , Neoplasias Ováricas/genética , Proteínas Represoras/biosíntesis , Factores de Transcripción/biosíntesis , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Células Epiteliales/patología , Epitelio/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Proteínas de Homeodominio/genética , Humanos , MicroARNs/genética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Proteínas Represoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc , Homeobox 1 de Unión a la E-Box con Dedos de ZincRESUMEN
We used massively parallel pyrosequencing to discover and characterize microRNAs (miRNAs) expressed in human embryonic stem cells (hESC). Sequencing of small RNA cDNA libraries derived from undifferentiated hESC and from isogenic differentiating cultures yielded a total of 425,505 high-quality sequence reads. A custom data analysis pipeline delineated expression profiles for 191 previously annotated miRNAs, 13 novel miRNAs, and 56 candidate miRNAs. Further characterization of a subset of the novel miRNAs in Dicer-knockdown hESC demonstrated Dicer-dependent expression, providing additional validation of our results. A set of 14 miRNAs (9 known and 5 novel) was noted to be expressed in undifferentiated hESC and then strongly downregulated with differentiation. Functional annotation analysis of predicted targets of these miRNAs and comparison with a null model using non-hESC-expressed miRNAs identified statistically enriched functional categories, including chromatin remodeling and lineage-specific differentiation annotations. Finally, integration of our data with genome-wide chromatin immunoprecipitation data on OCT4, SOX2, and NANOG binding sites implicates these transcription factors in the regulation of nine of the novel/candidate miRNAs identified here. Comparison of our results with those of recent deep sequencing studies in mouse and human ESC shows that most of the novel/candidate miRNAs found here were not identified in the other studies. The data indicate that hESC express a larger complement of miRNAs than previously appreciated, and they provide a resource for additional studies of miRNA regulation of hESC physiology. Disclosure of potential conflicts of interest is found at the end of this article.
Asunto(s)
Células Madre Embrionarias/metabolismo , Perfilación de la Expresión Génica , Biblioteca de Genes , MicroARNs/genética , Análisis de Secuencia de ARN , Secuencia de Bases , Diferenciación Celular , Línea Celular , Bases de Datos Genéticas , Células Madre Embrionarias/citología , Células Madre Embrionarias/enzimología , Etiquetas de Secuencia Expresada , Regulación del Desarrollo de la Expresión Génica , Humanos , MicroARNs/química , Datos de Secuencia Molecular , Células Madre Multipotentes/citología , Células Madre Multipotentes/metabolismo , Conformación de Ácido Nucleico , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleasa III/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Epithelial-to-mesenchymal transition (EMT) is a key process associated with tumor progression and metastasis. To define molecular features associated with EMT states, we undertook an integrative approach combining mRNA, miRNA, DNA methylation, and proteomic profiles of 38 cell populations representative of the genomic heterogeneity in lung adenocarcinoma. The resulting data were integrated with functional profiles consisting of cell invasiveness, adhesion, and motility. A subset of cell lines that were readily defined as epithelial or mesenchymal based on their morphology and E-cadherin and vimentin expression elicited distinctive molecular signatures. Other cell populations displayed intermediate/hybrid states of EMT, with mixed epithelial and mesenchymal characteristics. A dominant proteomic feature of aggressive hybrid cell lines was upregulation of cytoskeletal and actin-binding proteins, a signature shared with mesenchymal cell lines. Cytoskeletal reorganization preceded loss of E-cadherin in epithelial cells in which EMT was induced by TGFß. A set of transcripts corresponding to the mesenchymal protein signature enriched in cytoskeletal proteins was found to be predictive of survival in independent datasets of lung adenocarcinomas. Our findings point to an association between cytoskeletal and actin-binding proteins, a mesenchymal or hybrid EMT phenotype and invasive properties of lung adenocarcinomas.