The Analysis Of Prp And The Mutants Using Rnai Technology

Transmissible spongiform encephalopathies (TSEs), prion diseases, are rare degenerative neurological disorders that afflict human beings, including Creutzfeldt-Jacob disease (CJD), Gerstmann-Straussler-Scheinker syndrome (GSS), Kuru, fatal familial insomnia (FFI), and sheep and goat scrapie, bovine spongiform encephalopathy (BSE), elk chronic wasting disease (CWD) and other TSEs. Based on the important role of PrPc in the prion diseases, one of the current treatment strategies is to reduce the level of the normal protein PrPC and decrease the duplication of prions in order to prolong the patient's life. RNAi is highly conservative, sequence-specific technology in gene silencing mechanism, which could degrade the homologous messenger RNA. It has shown the potential of treatment in several neurodegenerative diseases, whose key point is the expression of the double-strand RNA in vivo.In this report, based on the analyses of the known sequences of human PrP, we constructed two small interfering RNA (siRNA) duplexes targeting the segments of aa (amino acids) 108-114 (Ri2) and aa 171-177 (Ri3). Western blot results revealed that two PrP-specific siRNAs could effectively knock down the levels of either endogenous PrP in human neuroblastoma SHSY5Y cells or recombinant PrP transfected with the plasmid expressing wide-type human PrP in human embryonic kidney (HEK) 293T cells. Meanwhile, two siRNAs also showed obvious effect on the reduction of the expressions of PrP-PG9 and PrP-PG12 that were familial Creutzfeldt-Jacob disease (CJD)-associated PrP mutants with four and seven extra octarepeats insertion in the cells transfected with respective expressing plasmids. Knocking down the levels of wild-type PrP by Ri2 and Ri3 did not change the cell growth capacities, but the MTT tests show that siRNAs significantly decreased the cell resistances against the challenge of Cu2+. Co-expressions of Ri2 and Ri3 partially antagonized the cytotoxicities caused by expressing PrP-PG9 and -PG12 in those two cell lines. Moreover, the rescuing effectiveness of PrP siRNAs showed clearly time-related feature that more efficient influences on antagonizing the cytotoxicities of fCJD-associated PrP mutants occurred at the early stage after transfection.PrPC is believed to be responsible for the metabolism of copper, lipid intake and signal transduction. PrPc could interact and co-localize with tublin in vivo and transport along the microtubule. The cytoPrP combined with tublin could inhibit the polymerization of microtubule, resulting in the dysfunction of mitosis and apoptosis. Western blot results showed that the expression level of cellular tublin declined obviously when PrP-PG9 or PrP-PG12 were introduced in the 293T cell lines. Co-transfection of the small interference RNA plasmid Ri3 obviously blocked the decreased activities of various PrP mutants on cellular tubulin. Moreover, we found that transfection of the Ri3 plasmid 12 h after transfections of CytoPrP, PrP-PG9 and PrP-PG12 in the cells could rescued the levels of cellular tublin to the same level as the control, but failed to recover the tubulin levels when transfection of the Ri3 plasmid 24 h after introductions of PrP mutants. The results of the confocal microscopy of cellular structures of microtubules displayed that both PrP-PG9 and PrP-PG12 inhibited the polymerization of microtubules. In line with above observations, introduction of the Ri3 plasmid 12 h after transfections of CytoPrP, PrP-PG9 and PrP-PG12 in the 293T cells could rescue the cellular structures of microtubules. It indicates that transfection of Ri3 timely is able to antagonize the cytotoxicity effects of PrP mutants, at least partially via blocking the destructive activities of PrP mutants on cellular microtubules.