Establishment and characterization of Prnp knock-down neuroblastoma cells using dual microRNA-mediated RNA interference

摘要

Prion diseases are fatal transmissible neurodegenerative disorders. In the pathogenesis of the disease, the cellular prion protein (PrP C) is required for replication of abnormal prion (PrP Sc), which results in accumulation of PrP Sc. Although there have been extensive studies using Pmp knockout systems, the normal function of PrP C remains ambiguous. Compared with conventional germline knockout technologies and transient naked siRNA-dependent knockdown systems, newly constructed durable chained-miRNA could provide a cell culture model that is closer to the disease status and easier to achieve with no detrimental sequelae. The selective silencing of a target gene by RNA interference (RNAi) is a powerful approach to investigate the unknown function of genes in vitro and in vivo. To reduce PrP C expression, a novel dual targeting-microRNA (miRdual) was constructed. The miRdual, which targets N- and C-termini of Pmp simultaneously, more effectively suppressed PrP C expression compared with conventional single site targeting. Furthermore, to investigate the cellular change following PrP C depletion, gene expression analysis of PrP C interacting and/or associating genes and several assays including proliferation, viability and apoptosis were performed. The transcripts 670460F02Rik and Plk3, Ppp2r2b and Csnk2a1 increase in abundance and are reported to be involved in cell proliferation and mitochondrial-mediated apoptosis. Dual-targeting RNAi with miRdual against Pmp will be useful for analyzing the physiological function of PrP C in neuronal cell lines and may provide a potential therapeutic intervention for prion diseases in the future.