RESUMO
Higher-grade gliomas are distinguished by increased vascular endothelial cell proliferation and peritumoral edema. These are thought to be instigated by vascular endothelial growth factor, which in turn is regulated by cellular oxygen tension. Hypoxia inducible factor-1alpha (HIF-1alpha) is a main responder to intracellular hypoxia and is overexpressed in many human cancers, including gliomas. Here we present methods for investigating the role of HIF-1alpha in glioma growth in vivo and in vitro using RNA interference in U251, U87, and U373 glioma cells.
Assuntos
Neoplasias Encefálicas/terapia , Glioma/terapia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Interferência de RNA , RNA Interferente Pequeno/uso terapêutico , Animais , Western Blotting , Neoplasias Encefálicas/genética , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica/efeitos dos fármacos , Terapia Genética/métodos , Glioma/genética , Transportador de Glucose Tipo 1/metabolismo , Humanos , Hipóxia , Técnicas Imunoenzimáticas , Camundongos , Camundongos Nus , Reação em Cadeia da Polimerase , Transfecção , Ubiquitina-Proteína Ligases/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
A RNA interference (RNAi) like mechanism is involved in elimination of thousands of DNA segments from the developing somatic macronucleus of Tetrahymena, yet how specific internal eliminated sequences (IESs) are recognized remains to be fully elucidated. To define requirements for DNA rearrangement, we performed mutagenesis of the M element, a well-studied IES. While sequences within the macronucleus-retained DNA are known to determine the excision boundaries, we show that sequences internal to these boundaries are required to promote this IES's rearrangement. However, this element does not contain any specific sequence required in cis as removal of its entire left or right side was insufficient to abolish all rearrangement. Instead, rearrangement efficiency correlated with the overall size of the M element sequence within a given construct, with a lower limit of nearly 300 bp. Also, the observed minimal region necessary to epigenetically block excision supports this size limit. Truncated M element constructs that exhibited impaired rearrangement still showed full transcriptional activity, which suggests that their defect was due to inefficient recognition. This study indicates that IESs are targeted for elimination upon their recognition by homologous small RNAs and further supports the idea that DNA elimination is a RNAi-related mechanism involved in genome surveillance.