title: Establishment and characterization of Prnp knockdown neuroblastoma cells using dual microRNA-mediated RNA interference.
abstract: Prion diseases are fatal transmissible neurodegenerative disorders. In the pathogenesis of the disease, the cellular prion protein (PrPC) is required for replication of abnormal prion (PrPSc), which results in accumulation of PrPSc. Although there have been extensive studies using Prnp knockout systems, the normal function of PrPC 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 PrPC expression, a novel dual targeting-microRNA (miRdual) was constructed. The miRdual, which targets N- and C- termini of Prnp simultaneously, more effectively suppressed PrPC expression compared with conventional single site targeting. Furthermore, to investigate the cellular change following PrPC depletion, gene expression analysis of PrPC 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 Prnp will be useful for analyzing the physiological function of PrPC in neuronal cell lines and may provide a potential therapeutic intervention for prion diseases in the future.
authors.affiliation: Department of Infectious Diseases, College of Veterinary Medicine, KRF Zoonotic Disease Priority Research Institute and BK21 Program for Veterinary Science, Seoul National University, Seoul, Korea.
authors.forename: Sang-Gyun
authors.initials: SG
authors.lastname: Kang
authors.forename: Yu-Mi
authors.initials: YM
authors.lastname: Roh
authors.forename: Agnes
authors.initials: A
authors.lastname: Lau
authors.forename: David
authors.initials: D
authors.lastname: Westaway
authors.forename: Debbie
authors.initials: D
authors.lastname: McKenzie
authors.forename: Judd
authors.initials: J
authors.lastname: Aiken
authors.forename: Yong-Sun
authors.initials: YS
authors.lastname: Kim
authors.forename: Han Sang
authors.initials: HS
authors.lastname: Yoo
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics / Q000502:physiology; D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002470:Cell Survival / Q000235:genetics / Q000502:physiology; D051379:Mice; D035683:MicroRNAs / Q000235:genetics*; D009447:Neuroblastoma / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D000072002:Prion Proteins; D011328:Prions / Q000235:genetics* / Q000378:metabolism*; D034622:RNA Interference / Q000502:physiology*
chemical_list: D035683:MicroRNAs; D000072002:Prion Proteins; D011328:Prions; C512657:Prnp protein, mouse