title: Knockdown of prion protein (PrP) by RNA interference weakens the protective activity of wild-type PrP against copper ion and antagonizes the cytotoxicity of fCJD-associated PrP mutants in cultured cells.
abstract: Development of the pathogenesis of transmissible spongiform encephalopathies (TSEs) requires the presence of both the normal host prion protein (PrPC) and the abnormal pathological proteinase-K resistant isoform (PrPSc). Reduction of PrPC levels has been shown to extend survival time after prion infection. In this report, based on analysis of the known sequences of human PrP, we constructed two small interfering RNA (siRNA) duplexes targeting the segments of amino acids (aa) 108-114 (Ri2) and aa 171-177 (Ri3). Western blot analysis results revealed that these PrP-specific siRNAs could effectively knock down the levels of either endogenous PrP in human neuroblastoma SHSY-5Y cells or recombinant PrP transfected with the plasmid expressing the full-length human PrP in human embryonic kidney (HEK) 293T cells. Meanwhile, the two siRNAs also showed a significant effect on the reduction of the expression of the PrP-PG9 and PrP-PG12 familial Creutzfeldt-Jakob disease (CJD)-associated PrP mutants with four and seven extra octarepeats, in the cells transfected with the respective expression plasmids. MTT tests identified that knockdown of wild-type PrP by Ri2 and Ri3 did not change the cell growth capacities, but significantly decreased the cell resistances against the challenge of Cu2+. Co-expression of Ri2 and Ri3 partially antagonized the cytotoxicity caused by expressing PrP-PG9 and PrP-PG12 in the two cell lines. Moreover, the rescuing effectiveness of PrP siRNAs was time-related, with the more efficient antagonism of the cytotoxicity of fCJD-associated PrP mutants occurring at the early stages after transfection. The data shown here provide useful clues for seeking potential therapeutic tools for prion diseases.
authors.affiliation: State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, PR China.
authors.forename: Zhao-Yun
authors.initials: ZY
authors.lastname: Wang
authors.forename: Chan
authors.initials: C
authors.lastname: Tian
authors.forename: Yuan-Yuan
authors.initials: YY
authors.lastname: Jing
authors.forename: Han-Shi
authors.initials: HS
authors.lastname: Gong
authors.forename: Yan
authors.initials: Y
authors.lastname: Guo
authors.forename: Qi
authors.initials: Q
authors.lastname: Shi
authors.forename: Cao
authors.initials: C
authors.lastname: Chen
authors.forename: Shu-Ying
authors.initials: SY
authors.lastname: Zhu
authors.forename: Xiao-Ping
authors.initials: XP
authors.lastname: Dong
mesh_terms: D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D002470:Cell Survival; D003300:Copper / Q000633:toxicity*; D007562:Creutzfeldt-Jakob Syndrome / Q000235:genetics; D019286:Endopeptidase K / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D007477:Ions / Q000378:metabolism*; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009447:Neuroblastoma / Q000378:metabolism; D010957:Plasmids; D018621:PrPC Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011328:Prions / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA
chemical_list: D007477:Ions; D050505:Mutant Proteins; D018621:PrPC Proteins; D011328:Prions; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D003300:Copper; D019286:Endopeptidase K