RESUMEN
We developed an array-based subtractive hybridization system for one-step high-throughput subtraction. We printed subtractor RNA up to 10.000 times obtaining an excellent contact surface using a little amount of RNA. During hybridization cDNA, common to subtractor and target samples, remains attached to slide immobilized RNA, leaving free in solution target specific cDNA which after retrieval is cloned.
Asunto(s)
Clonación Molecular/métodos , ADN Complementario/metabolismo , Análisis por Micromatrices/métodos , Hibridación de Ácido Nucleico/métodos , ARN/metabolismoRESUMEN
MicroRNAs are aberrantly expressed in many cancers and can exert tumour-suppressive or oncogenic functions. As oncomirs promote growth of cancer cells and support survival during chemotherapy, thus microRNA-silencing therapies could be a valuable approach to be associated with anticancer drugs and chemotherapy treatments. miR-155 microRNA was found overexpressed in different types of cancer, such as leukaemias (PML, B-cell lymphomas), lung cancer and glioblastoma. GABA-A receptor downregulation was found correlated with glioma grading, with decreasing levels associated with higher grade of malignancies. A relationship between knock-down of miR-155 and re-expression of GABRA 1 protein in vivo was recently individuated. This finding has implication on the effectiveness of RNA-silencing approaches against miR-155 with the scope to control proliferation and signalling pathways regulated by GABA-A receptor. Applying microRNAs for treatment of brain tumours poses several problems, and fields to be solved are mainly the passage of the brain-blood barrier and the targeted delivery to specific cell types. Glioblastoma multiforme cells bud off microvesicles that deliver cytoplasmic contents to nearby cells. Thus, the exploitation of these mechanisms to deliver antagomir therapeutics targeting microvescicles in the brain could take the lead in the near future in the treatment for brain cancers in substitution of invasive surgical intervention.
Asunto(s)
Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , MicroARNs/metabolismo , Oligonucleótidos Antisentido/uso terapéutico , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Humanos , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , Interferencia de ARN , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismoRESUMEN
An altered expression of microRNAs (miRNAs) contributes both to the development of cancer and to the progression of the disease. Malignant tumours and tumour cell lines have widespread deregulated expressions of miRNAs compared to normal tissues. In this study, we investigated the expression profiles of 340 mammalian miRNAs in 93 cases of multiform glioblastoma (primary and secondary glioblastoma tumours), by means of DNA microarrays. We show that the expression profiles of 10 miRNAs can distinguish primary from secondary glioblastoma types. Moreover, we found elevated miR-155 levels in primary and secondary glioblastoma tissues as well as in glioblastoma primary cultures. We hypothesised that γ-aminobutyric acid A receptor 1 (GABRA1) is a miR-155 target, and studied the correlation between miR-155 up-regulation and the GABRA1 protein in cultured glioblastoma cells by miRNA silencing. We show that a decrease in miR-155 expression to normal levels restores the expression of GABRA1, making glioblastoma cells sensitive to signals that inhibit cell proliferation mediated by GABRA1. In conclusion, the expression patterns of different miRNAs characterise primary and secondary glioblastomas. The aberrant overexpression of miR-155 contributes to the malignant phenotype of glioblastoma cells removing growth inhibition.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Regulación Neoplásica de la Expresión Génica , Glioblastoma/metabolismo , MicroARNs/genética , Receptores de GABA-A/genética , Regulación hacia Arriba , Adulto , Anciano , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/patología , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Análisis por Conglomerados , Diagnóstico Diferencial , Femenino , Perfilación de la Expresión Génica , Glioblastoma/diagnóstico , Glioblastoma/secundario , Humanos , Masculino , MicroARNs/metabolismo , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de GABA-A/metabolismo , Células Tumorales Cultivadas , Adulto JovenRESUMEN
BACKGROUND: The importance of non-coding RNAs (ncRNAs) as fine regulators of eukaryotic gene expression has emerged by several studies focusing on microRNAs (miRNAs). miRNAs represent a newly discovered family of non coding-RNAs. They are thought to be crucial players of human hematopoiesis and related tumorigenesis and to represent a potential tool to detect the early stages of cancer. More recently, the expression regulation of numerous long ncRNAs has been linked to cell growth, differentiation and cancer although the molecular mechanism of their function is still unknown.NB4 cells are promyelocytic cells that can be induced to differentiation upon retinoic acid (ATRA) treatment and represent a feasible model to study changes of non coding RNAs expression between cancer cells and their terminally differentiated counterpart. FINDINGS: we screened, by microarray analysis, the expression of 243 miRNAs and 492 human genes transcribing for putative long ncRNAs different from miRNAs in NB4 cells before and after ATRA induced differentiation. Our data show that 8 miRNAs, and 58 long ncRNAs were deregulated by ATRA induced NB4 differentiation. CONCLUSION: our data suggest that ATRA-induced differentiation lead to deregulation of a large number of the ncRNAs that can play regulatory roles in both tumorigenesis and differentiation.