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.

Figure 1

Schematic features of miR cassettes and knockdown efficacy in N2a cells. (A) Each miR cassette in pcDNA6.2-GW/EmGFP vector. (B) Western blot of N2a cells transfected with each miR expression construct. (C) Quantitation of PrP^C expression. When miRdual was introduced into the cells, PrP^C expression was the most efficiently decreased as compared with wild-type cells (*p < 0.05, **P <0.01).

Figure 2

Transfection of miR cassettes and Prnp knockdown efficacy at mRNA and protein level. (A) PCR of genomic DNA shows miR cassette-specific amplicons. Lane L, 100 bp DNA ladder; lane 1, expression vector with miRdual cassette; lane 2, expression vector with miRscr; lane 3, wild-type cells; lane 4, N2amiRdual; lane 5, N2amiRscr; lane 6, no template. (B) qPCR analysis of PrP^C transcript abundance showing knockdown efficiency of miRdual in mRNA levels. (C) Western blot analysis of PrP^C protein abundance. Top part is the western blot and bottom panel is densitometric evaluation. dual1 and dual2 are N2amiRdual cell clones (*p < 0.05, **p < 0.01).

Figure 3

Quantitative PCR analysis of PrP^C interacting and/or associated molecules. 6720460F02Rik, Plk3, Ppp2r2b, Csnk2a1 transcripts were upregulated in prion knockdown N2amiRdual cells compared to wild-type N2a cells. The increased Mpg transcripts in both N2amiRdual and N2amiRscr seemed to have no correlation with decreased PrP^C (*p < 0.05, **p < 0.01).

Figure 4

Cell proliferation, viability and GFP expression test. (A) Viability and proliferation of N2a cells stably transformed with miRdual or miRscr (*p < 0.05). (B) Confocal microscopy images of clonal expanded cells. N2amiRdual and N2amiRscr showed green fluorescent signals. Cell nuclei were stained with DAPI (blue). Photos were taken at 20 doubling passages. GFP signals stably expressed over 135 doubling passages. Scale bar, 50 um.

Figure 5

Apoptotic resistance to serum deprivation. Cells were cultured under conditions of serum deprivation and collected at indicated time points. (A) Detection of Drp1 expression levels analyzed by densitometric analysis of western blot. (B) Cytochrome C expression in cytosolic fractions detected by immunoassay. (C) Analysis of caspase 3 activity calculated by comparison with the free pNA. dual, N2amiRdual; scr, N2amiRscr; wt, miR non-treated wild-type N2a (*p < 0.05, **p < 0.01).

Figure 6

Detection of apoptotic cells following serum deprivation using Annexin V-Cy3 assay. Cells were collected at the indicated time points and 5 × 10⁵ cell suspensions were treated with Annexin V. The proportion of cells showing apoptotic change was increased in N2amiRdual whereas most of cells showed the resistance against apoptosis stimulation in N2amiRscr and wild-type N2a cells (*p < 0.05).

Figure 7

Downregulation pattern of PrP^C in RK13-moPrnp cells harvested at different confluences. RK13-moPrnp cells were lentiviral-transduced with miR expression cassettes and the relative abundances of PrP^C and GFP processed were determined by western blot (*p < 0.05, **p < 0.01).