RESUMO
3D models are increasingly used to study mechanisms driving tumor progression and mimicking in vitro processes such as invasion and migration. However, there is a need to establish more protocols based on 3D culture systems that allow for downstream molecular biology investigations. Materials & methods: Here we present a method for optimal RNA extraction from highly aggressive primary glioma cells invading into Matrigel. The method has been established by comparing previously reported protocols, available commercial kits and optimizing specific steps for matrix dissociation, RNA separation and purification. Results and conclusion: The protocol allows RNA extraction from cells embedded into Matrigel, with optimal yield, purity and integrity suitable for subsequent sequencing analysis of both high and low molecular weight RNA.
Assuntos
Colágeno , Glioma/patologia , Laminina , Invasividade Neoplásica , Proteoglicanas , RNA , Linhagem Celular Tumoral , Movimento Celular , Combinação de Medicamentos , Humanos , RNA/isolamento & purificaçãoRESUMO
Ewing sarcoma (EwS) is an aggressive tumor that affects adolescents and young adults. EwS is defined by a chromosomal translocation, EWSR1-FLI1 being the most common, that causes genome reprogramming through remodeling of enhancers. Here, we describe an unexpected function of RING1B, which is highly expressed in EwS. While retaining its repressive activity at Polycomb developmental regulated genes, RING1B colocalizes with EWSR1-FLI1 at active enhancers. We demonstrate that RING1B is necessary for the expression of key EWSR1-FLI1 targets by facilitating oncogene recruitment to their enhancers. Knockdown of RING1B impairs growth of tumor xenografts and expression of genes regulated by EWSR1-FLI1 bound enhancers. Pharmacological inhibition of AURKB with AZD1152 increases H2Aub levels causing down-regulation of RING1B/EWSR1-FLI1 common targets. Our findings demonstrate that RING1B is a critical modulator of EWSR1-FLI1-induced chromatin remodeling, and its inhibition is a potential therapeutic strategy for the treatment of these tumors.