RESUMEN
MicroRNAs are a class of sophisticated regulators of gene expression, acting as post-transcriptional inhibitors that recognize their target mRNAs through base pairing with short regions along the 3'UTRs. Several microRNAs are tissue specific, suggesting a specialized role in tissue differentiation or maintenance, and quite a few are critically involved in tumorigenesis. We studied miR-128, a brain-enriched microRNA, in retinoic acid-differentiated neuroblastoma cells, and we found that this microRNA is up-regulated in treated cells, where it down-modulates the expression of two proteins involved in the migratory potential of neural cells: Reelin and DCX. Consistently, miR-128 ectopic overexpression suppressed Reelin and DCX, whereas the LNA antisense-mediated miR-128 knockdown caused the two proteins to increase. Ectopic miR-128 overexpression reduced neuroblastoma cell motility and invasiveness, and impaired cell growth. Finally, the analysis of a small series of primary human neuroblastomas showed an association between high levels of miR-128 expression and favorable features, such as favorable Shimada category or very young age at diagnosis. Thus, we provide evidence for a role for miR-128 in the molecular events modulating neuroblastoma progression and aggressiveness.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Regulación de la Expresión Génica/fisiología , MicroARNs/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuroblastoma/tratamiento farmacológico , Neuropéptidos/metabolismo , Serina Endopeptidasas/metabolismo , Secuencia de Bases , Moléculas de Adhesión Celular Neuronal/genética , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Proteínas de la Matriz Extracelular/genética , Humanos , MicroARNs/genética , Proteínas Asociadas a Microtúbulos/genética , Datos de Secuencia Molecular , Invasividad Neoplásica , Proteínas del Tejido Nervioso/genética , Neuroblastoma/metabolismo , Neuronas/efectos de los fármacos , Neuronas/fisiología , Neuropéptidos/genética , Proteína Reelina , Serina Endopeptidasas/genética , Tretinoina/farmacologíaRESUMEN
Angiogenesis is required for the development and biologic progression of glioblastoma multiform (GBM), which is the most malignant infiltrative astrocytoma. Vascular endothelial growth factor (VEGF) plays a predominant role in the increased vascularity and endothelial cell proliferation in GBMs driven by the expression of pro-angiogenic cytokines. In this study, we employed a vector-encoded VEGF siRNA to impair VEGF secretion from U87 human glioblastoma cells. The direct intra-tumor injection of a siRNA-encoding plasmid complexed with linear polyethylenimine (PEI) efficiently reduced the vascularization of treated tumors in xenografts established in SCID mice by subcutaneous inoculation of U87 cells, but was not able to reduce tumor growth. We then sought to strengthen the in vivo action of our siRNA by coupling it to a well known direct antiangiogenic agent, mouse interleukin 4 (mIL4). We infected U87 cells with a retroviral vector coexpressing the VEGF siRNA and mIL4 and produced stable cell lines that we used for an in vivo experiment of subcutaneous injection in SCID mice. In this setting, the concomitant expression of mIL4 and siRNA totally abolished the growth of subcutaneous tumors. These results suggest that our retroviral vector might be employed as a potential tool in future antiangiogenic gene therapy trials for glioblastoma.
Asunto(s)
Neoplasias Encefálicas/terapia , Terapia Genética , Glioblastoma/terapia , Interleucina-4/uso terapéutico , Neovascularización Patológica/terapia , Factor A de Crecimiento Endotelial Vascular/genética , Animales , Neoplasias Encefálicas/irrigación sanguínea , Neoplasias Encefálicas/tratamiento farmacológico , Terapia Combinada , Regulación hacia Abajo , Glioblastoma/irrigación sanguínea , Glioblastoma/tratamiento farmacológico , Humanos , Ratones , Ratones SCID , Neovascularización Patológica/tratamiento farmacológico , Plásmidos/administración & dosificación , Plásmidos/genética , ARN Interferente Pequeño/genética , Retroviridae/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Neuroleukin (NLK) is a multifunctional protein, involved in neuronal growth, glucose metabolism, cell motility, and differentiation. Expressed in the brain, it supports the growth of embryonic spinal, skeletal motor, and sensory neurons. We have previously demonstrated that NLK is up-regulated in the brain during Huntington's disease (HD), a neurodegenerative disorder caused by the expansion of CAG trinucleotide repeats. In order to study the biological role of NLK, we have generated an inducible rat pheochromocytoma PC12 cell line in which the expression of NLK is selectively down-regulated by antisense strategy. We show here that the block of NLK commits PC12 cells to caspase-dependent apoptosis. This priming effect elicited by NLK inhibition is independent from the differentiation state of the neuronal cells. These results suggest a general protective role of NLK in the control of cell death in neuronal cells.
Asunto(s)
Apoptosis , Caspasas/metabolismo , Glucosa-6-Fosfato Isomerasa/antagonistas & inhibidores , Glucosa-6-Fosfato Isomerasa/fisiología , Neuronas/metabolismo , Clorometilcetonas de Aminoácidos/farmacología , Animales , Western Blotting , Muerte Celular , ADN Complementario/metabolismo , Regulación hacia Abajo , Electroforesis en Gel de Poliacrilamida , Citometría de Flujo , Vectores Genéticos , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Oligonucleótidos Antisentido/farmacología , Células PC12 , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
Hepatitis C virus (HCV) has evolved complex strategies to evade host immune responses and establish chronic infection. The only treatment available for HCV infections, alpha interferon (IFN-alpha), is effective in a limited percentage of patients. The mechanisms by which IFN-alpha interferes with the HCV life cycle and the reasons for limited effectiveness of IFN-alpha therapy have not yet been fully elucidated. Using a cell-based HCV replication system and specific kinase inhibitors, we examined the role played by various signaling pathways in the IFN-alpha-mediated HCV clearance. We reported that conventional protein kinase C (cPKC) activity is important for the effectiveness of IFN-alpha treatment. In cells treated with a cPKC-specific inhibitor, IFN-alpha failed to induce an efficient HCV RNA degradation. The lack of cPKC activity leads to a broad reduction of IFN-alpha-stimulated gene expression due to a significant impairment of STAT1 and STAT3 tyrosine phosphorylation. Thus, modulation of cPKC function by either host or viral factors could influence the positive outcome of IFN-alpha-mediated antiviral therapies.