RESUMO
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.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Regulação da Expressão Gênica/fisiologia , MicroRNAs/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuroblastoma/tratamento farmacológico , Neuropeptídeos/metabolismo , Serina Endopeptidases/metabolismo , Sequência de Bases , Moléculas de Adesão Celular Neuronais/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proteínas do Domínio Duplacortina , Proteína Duplacortina , Proteínas da Matriz Extracelular/genética , Humanos , MicroRNAs/genética , Proteínas Associadas aos Microtúbulos/genética , Dados de Sequência Molecular , Invasividade Neoplásica , Proteínas do Tecido Nervoso/genética , Neuroblastoma/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Neuropeptídeos/genética , Proteína Reelina , Serina Endopeptidases/genética , Tretinoína/farmacologiaRESUMO
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.