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.
Prion. 2011 Apr-Jun;5(2):93-102. doi: 10.4161/pri.5.2.15621. Epub 2011 Apr 1.
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.
朊病毒病是致命的可传播神经退行性疾病。在疾病发病机制中，细胞朊蛋白（PrPC）是复制异常朊病毒（PrPSc）所必需的，这导致 PrPSc 的积累。尽管已经使用 Prnp 敲除系统进行了广泛的研究，但 PrPC 的正常功能仍不清楚。与传统的种系敲除技术和瞬时裸露 siRNA 依赖性敲低系统相比，新构建的持久链式-miRNA 可以提供更接近疾病状态的细胞培养模型，并且更容易实现，没有不良后遗症。通过 RNA 干扰（RNAi）对靶基因进行选择性沉默是一种强大的方法，可以在体外和体内研究基因的未知功能。为了降低 PrPC 的表达，构建了一种新型的双重靶向 miRNA（miRdual）。miRdual 同时靶向 Prnp 的 N 和 C 末端，与传统的单点靶向相比，更有效地抑制了 PrPC 的表达。此外，为了研究 PrPC 耗竭后细胞的变化，对 PrPC 相互作用和/或相关基因的基因表达分析以及包括增殖、活力和凋亡在内的几种测定进行了研究。转录本 670460F02Rik 和 Plk3、Ppp2r2b 和 Csnk2a1 的丰度增加，并被报道参与细胞增殖和线粒体介导的细胞凋亡。针对 Prnp 的 miRdual 的双重靶向 RNAi 将有助于分析神经元细胞系中 PrPC 的生理功能，并可能为未来的朊病毒病提供潜在的治疗干预措施。
