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ISSN : 2287-7991(Print)
ISSN : 2287-8009(Online)
Journal of the Preventive Veterinary Medicine Vol.36 No.4 pp.186-195
DOI :

Gene expression profile of a persistently chronic wasting disease (CWD) prion-infected RK13 cell line

Yoon-Hee Lee, Hyo-Jin Kim*, Won-Yong Lee*, Min-Jeong Kim, Dong-Seob Tark, In-Soo Cho, Hyun-Joo Sohn
Animal, Plant and Fishery Quarantine Inspection and Agency
Received 30 October 2012, revised 7 December 2012, accepted 11 December 2012.

Abstract

Chronic wasting disease (CWD) is a neurodegenerative disorder in cervids and a member of the transmissiblespongiform encephalopathies (TSEs), also known as prion diseases. We previously generated a persistently CWD prioninfected RK13 cell line (RKC1-11) using elk PrPC expressing cells (elkRK13) that were generated with the lentiviral expressionsystem. To investigate the differentially expressed (DE) genes involved in prion infection at the cellular level, we performedmicroarray analysis and identified the DE genes between CWD-infected (RKC1-11) and non-infected cells (elkRK13).Collectively, 88 genes were found to be differentially expressed (42 genes upregulated > 2-fold; 46 genes downregulated <0.5-fold); additionally, 10 up- and 8 downregulated genes agreed with the results of the qRT-PCR. Among these genes, wechose 8 DE genes associated with cell growth, signal transduction, transport, immune response and apoptosis based on genefunction analysis for further analysis. The expression of the selected genes was further validated using an animal model innormal- and CWD-infected TgElk mice showing clinical signs at 185 dpi. These identified DE genes in both the in vitro andin vivo model could help in understanding the diagnosis and pathogenesis of prion diseases.

Prion,RK13,elk,CWD,qRT-PCR
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INTRODUCTION

CWD (Chronic Wasting Disease) is a neurodegenerative disorder in cervids and a member of the prion diseases known as the transmissible spongiform encephalopathies (TSEs): other prion diseases include Scrapie in sheep and goats, Bovine Spongiform Encephalopathy (BSE) in cattle, and Creutzfeldt-Jakob Disease (CJD) in humans. TSEs are characterized by abnormal prion proteins (PrPSc) acting as infectious agents, which are generated by posttranslational modifications of normal prion proteins (PrPC) that accumulate in the brain and lead to diseases [10, 15, 21, 28, 29]. A major event in the pathogenesis of TSE is the structural conversion of PrPC to a partially PK resistant PrPSc by an unknown process [1]. CWD has been recognized as an important prion disease in native North America deer and Rocky mountain elk [29] and the only one found in freeranging species. Indeed, TSEs have not been found in any animal species in the Republic of Korea except for CWD, which is reported to have originated from imported elk from Canada [27]. National CWD surveillance has been conducted since 2001, and subsequent cases were found in farmed elk in 2004 and 2005 [13]. More recently, CWD cases were detected in farmed elk, Sika deer, red deer, and crossbred Sika and red deer in 2010 (unpublished data). A number of studies using microarray tools have shown TSE-associated changes of gene expression in brain tissues of rodent adapted models of prion diseases including CJD, scrapie and BSE [2, 4, 8, 23, 26, 30, 32]. These studies have represented alterations in multiple genes correlating with the pathogenesis of prion diseases including the regulation of apoptosis, stress response and metal ion homeostasis. We previously generated a persistently CWD prion infected RK13 cell line (RKC1-11) using elk PrPC expressing cells (elkRK13), which are rabbit kidney epithelial cells overexpressing exogenous elk PrPC, that was generated with the lentiviral expression system [12]. The microarray experiment for cervids was performed using bovine microarray analysis in different tissues of CWD-diseased elk due to insufficient cervid sequence data and genomic information [3]. As another approach, experiments for gene expression profiles in the RKC1-11 cell line were conducted to investigate the differentially expressed (DE) genes involved in CWD prion infection at the cellular level. In this study, we identified 88 DE genes between CWD-infected (RKC1-11) and non-infected cells (elkRK13) using rabbit whole genome microarray analysis. We selected 18 DE genes by analyzing the mouse gene function due to the lack of genome data for cervids and validated this data by performing qRT-PCR in the CWD-infected RK13 cell line. All together, the suggested 8 DE genes of the RKC1-11 cell line agreed with the results of the CWD-infected TgElk mice. The identified DE genes in both the CWD-infected cell line and TgElk mice from the present study could be potential candidates for biomarkers of CWD prion infection and help in understanding the pathogenesis of prion diseases. 

MATERIALS AND METHODS

Cell culture and animals

 The persistently CWD-infected RK13 cell line (RKC1-11) has been described by Kim et al. [12]. The experimental infection was performed by inoculating CWD-infected brain homogenates into Korean raised elk PrPC expressing cells (elkRK13) derived from the transformed cell line [11]. The cells were cultured in DMEM with a high glucose supplement, 10% FBS, penicillin (100 U/㎖), streptomycin (100 ㎍/ ㎖) and non-essential amino acids at 37℃ in a humidified 5% CO2  incubator. Cells were grown to confluence for RNA isolation and qRT-PCR analysis. TgElk mice over-expressing the elk prion protein [14] were kindly supplied by the New York State Institute for Basic Research in Developmental Disabilities, USA and inoculated intracranially with a pool of two CWD cases in 2001 (E190Y+229Y). Mice were sacrificed at the terminal stage of the disease, at 185 days post-infection (dpi). They exhibited clinical symptoms including affected gait, rough coat, hind limb paralysis, sticky eye and weight loss as previously described [16]. The brain of mice was frozen immediately in liquid nitrogen and stored at -80℃ until use.  

RNA isolation

Total RNA was extracted from the RK13 cell line using RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA was isolated from brain tissue homogenate by disruption in TRIzol reagent (Qiagen, Hilden, Germany). The quality of RNA was measured using an Agilent BioAnalyzerTM 2100 (Agilent technologies, CA, USA).  

Microarray

Each total RNA sample (200 ng) was labeled and amplified using the Low Input Quick Amp labeling kit (Agilent technologies, CA). The Cy3-labeled aRNAs were resuspended in 50 ㎕ of hybridization solution (Agilent technologies, CA). Afterwards, the labeled aRNAs were placed on the Agilent Rabbit whole genome 4×44K array (Agilent technologies, CA) and covered by a Gasket 4-plex slide (Agilent technologies, CA). The slides were hybridized for 17 hour at 65℃ in an oven. The hybridized slides were washed in 2×SSC, 0.1% SDS for 2 min, 1 × SSC for 3 min, and then 0.2 × SSC for 2 min at room temperature (RT). The slides were centrifuged at 3,000 rpm for 20 sec to dry. 

Data analysis

The arrays were analyzed using an Agilent scanner with associated software. Gene expression levels were calculated with Feature Extraction v10.7.3.1 (Agilent technologies, CA). Relative signal intensities for each gene were generated using the Robust Multi-Array Average algorithm. The data were processed based on the median polish normalization method using the GeneSpring GX 11.0 (Agilent technologies, CA). This normalization method aims to make the distribution of intensities for each array in a set of arrays the same. The normalized and log transformed intensity values were then analyzed using GeneSpring GX 11.0 (Agilent technologies, CA). The fold change filters included the requirement that the genes be present in at least 200% of the controls for upregulated genes and lower than 50% of the controls for downregulated genes. Hierarchical clustering data were clustered groups that behave similarly across experiments using GeneSpring GX 7.3.1 (Agilent technologies, CA). The clustering algorithm was the Euclidean distance and average linkage method.  

Quantitative real-time PCR analysis

First strand cDNA was prepared from the total RNA (1 ㎍) with Superscript Ш reverse transcriptase (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The expression levels of the target genes were evaluated with IDT PrimeTime qPCR Assays with ZEN double-quenched probes commercially available with optimized primer and probe concentrations (Integrated DNA technologies, CORALVILLE, IA, USA). Quantitative real-time PCR (qRT-PCR) was performed on an ABI Prism 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) in a 96-well optical plate with a final reaction volume of 25 ㎕. All reactions on each plate were prepared from a Gene Expression Master Mix protocol (ABI). For data analysis, the baseline and threshold for cycle threshold (Ct) calculation were set manually with the ABI Prism SDS v2.0.5 software. qRT-PCR analysis was made using the 2-ΔΔCT method and normalized to the GAPDH level as the endogenous control. Experiments were performed in triplicate. The primer probe sequences for the validation assays are shown in Table 4. According to this method, we show that the gene expression of the target genes for the infected samples was quantitatively measured relative to those of the RNA from the non-infected samples [18].  

RESULTS

In a previous study, we made elkRK13 cells, which showed that RK13 cells that express exogenous elk PrPC are susceptible to CWD prion infection and are permissive to persistent accumulation of PrPSc. We also established a persistently CWD-infected RK13 cell line (RKC1-11) [12]. Depending on specific cell type, it was reported that prion infection alters the cellular gene expression profile as a response to persistent prion propagation [8]. Here, we investigated the gene expression profile of the CWD prion infected RK13 cell line. RNA from the persistently infected cells (RKC1-11) and non-infected control cells (elkRK13) was isolated and subjected to microarray analysis. Microarray analysis identified 88 genes that were found to be differentially expressed (42 upregulated > 2-fold; 46 downregulated < 0.5-fold) between RKC1-11 and elkRK13 cells (Tables 1 and 2). Total RNA was extracted and cDNA was prepared from the RKC1-11 cells and elkRK13 control cells in exactly the same way as the microarray experiments. In each experiment, all the samples were processed in triplicates and each experiment was done in triplicates. To analyze the qRT-PCR data, we calculated a parameter defined as ΔΔCt [18], which indicates the changes in RNA transcription caused by the infected samples normalized to RNA transcription changes in the non-infected cells. A high ΔΔCt value, either negative or positive, indicates considerable changes in the transcription levels of a tested gene. A positive ΔΔCt value indicates downregulation of RNA transcription, whereas a negative ΔΔCt indicates upregulation of RNA transcription following CWD prion infection. As shown in Fig. 1, in the CWD-infected RK13 cell line, the expression levels for both upregulated (RLN1, CSN1S2, RFT-II, PRP, AGTR2, SLC10A2, TLR3, TRF, STK17A and EIF2AK2) and downregulated (SLC22A2, CYP2B5, SLC3A1, CKM, ITI-HC3, PAX6, INS and VTN) genes were not significantly altered in agreement with the microarray data (Tables 1 and 2). 

Table 1. Upregulated genes in the CWD-infected RK13 cell line

Table 2. Downregulated genes in the CWD-infected RK13 cell line

Fig. 1 Quantitative real-time PCR analysis of DE genes. Expression of the selected 10 upregulated (A) and 8 downregulated (B) genes, which were observed in the microarray results, were compared between elkRK13 and RKC1-11 cells described in Table 3. Gene expression of the CWD infected cells were quantitatively measured relative to the RNA levels from the control cells (elkRK13).

Among the DE genes in the RKC1-11 cell line, the top functional categories for the mouse species were involved in cellular function and maintenance, cell signaling and molecular transport, and immune response and apoptosis. We intended to find the gene expression response to CWD and regulated genes in specific pathways. We chose to analyze RLN1, AGTR2, TLR3, TRF, EIF2AK2, SLC22A2, PAX6 and VTN and also analyzed the behavior of PRP, the gene encoding for PrP, as suggested by the absence of a change in protein levels during prion infection. Therefore, we decided to use these candidate genes that could directly be involved in the infection process for further qRT-PCR experiments to confirm their differential expression. We compared the gene expression profiles of normal- and CWD-infected TgElk mice (N= 4) at 185 dpi which were at the clinical stage. As shown in the results of the qRT-PCR, the 5 upregulated genes in elkRK13 versus the data for the RKC1-11 cell line had a similar pattern when compared with the microarray analysis, while the 3 downregulated genes were significantly changed in the TgElk mouse at 185 dpi (Fig. 2). These data suggest that the differences in gene expression of SLC22A2, PAX6 and VTN genes found in the TgElk mouse (Fig. 2) were probably because of a direct association to the presence of PrPSc.  

Fig. 2 Expression of relaxin 1 (RLN1), angiotensin II receptor, type 2 (AGTR2), toll-like receptor 3 (TLR3), liver transferring (TRF), eukaryotic translation initiation factor 2-alpha kinase 2 (EIF2AK2) solute carrier family 22 (organic cation transporter), member 2 (SLC22A2), prion protein (PRP), paired box 6 (PAX6) and vitronectin (VTN) was studied in the TgElk mouse at 185 dpi. Results are expressed as the ratio of expression of the gene of interest relative to the GAPDH control gene in normal versus infected TgElk mice.

DISCUSSION

To identify the DE genes potentially involved in prion infection, we applied Agilent whole genome expression microarrays and described the gene expression profile of the CWD prion-infected RK13 (RKC1-11) cell line. We used not only elkRK13, but also the CWD-infected cells to reduce the variability of gene expression previously experienced when carrying out microarray experiments in infected cells or brains [1, 3, 8, 19, 23, 32]. Actually, we obtained only a very small number of genes showing variation compared with other studies. The microarray analysis data revealed a total of 88 genes that changed with at least a 2-fold upregulation and 0.5-fold downregulation in the CWD-infected RK13 cell line (Tables 1 and 2). Furthermore, the 18 selected genes were validated in a RKC1-11 cell line with qRT-PCR. The results were in good agreement with those of the microarray analysis. In this study, among the 6 upregulated genes observed between the qRT-PCR and microarray results in the CWD-infected cells, only 4 were known proteins including TLR3, TRF, EIF2AK2 and PRP and 2 were unknown proteins (RLN1 and AGTR2). This result was further confirmed in CWD-infected brain tissues with the qRT-PCR. The validation of the selected genes, which had previously been identified or unidentified, was confirmed in our study. Interestingly, it had been demonstrated that TLR3 was upregulated in the brains of mice who were infected with distinct strains of scrapie, ME7 and RML [32]. The elevated expression of the TLR3 gene involved in the immune response was detected in the qRT-PCR experiments shown in a previous and the present microarray study. These findings support the hypothesis that the enhancement of complement proteins and inflammatory factors and the associated glial activation in the diseased brain are important pathogenic events in prion disease. The other known upregulated gene TRF, which is an iron uptake protein, was identified in scrapie-infected hamster brains [5]. It was reported that prion diseases in affected human, mouse and hamster brains exhibit a state of iron deficiency in the presence of excess total brain iron [25]. In iron deficient conditions, TRF could be upregulated. Previous studies have shown an association of microglial activation and neurodegeneration with prion diseases. Apoptosis is a process common to neurodegenerative diseases or prion diseases that is induced by neuronal cell death and damage [6, 7]. We additionally observed an apoptosis associated gene, a key mediator of apoptotic pathways, eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2). An increase of EIF2AK2 in ME7-infected mice suggests one mechanism by which the increased interferon responsive gene expression would enhance disease progression [24, 31]. As previous reported, several S100 calcium binding proteins were identified, including S100A6, S100A4, S100A8 and S100A9, which showed increased expression in scrapie-infected mouse brains [32]. Alterations in S100 function have been implicated in cancer, Down syndrome and Alzheimer's disease [9, 33]. In addition, S100A4 production was significantly enhanced by relaxin (RLN) in human thyroid carcinoma cells [22]. Indeed, an increase in AT2 receptor expression has been observed under several pathological conditions including vascular injury, myocardial infarction, congestive heart failure, renal failure, brain ischemia, sciatic or optic nerve transsection and neurodegenerative diseases such as Huntington’s and Alzheimer’s disease [17, 20]. Therefore, the unknown genes of upregulation were RLN1 and AGTR2, related to signal transduction. As mentioned above, these genes should be considered for further examination in multiple biological processes involving prion diseases. Interestingly, in regards to downregulation, there were more differences than similarities among the functional classes of genes. Three genes, SLC22A2, PAX6 and VTN, were downregulated in the RKC1-11 cells while upregulated in the CWD- TgElk mice shown by the qRT-PCR experiments. The former information agreed with the microarray analysis results, the latter had different results. The data show that the interactions of individual regulated genes in specific pathways and biological factors could be involved in the biological process of converting PrPC to a partially PK resistant PrPSc. Therefore, this suggests that the distinct patterns of up- and downregulated gene expression in both the RKC1-11 cells and CWD-affected TgElk mice could reflect their unique biological response to prion infection and therapy, and aid in the selection of candidate biomarkers. The 5 upregulated and 3 downregulated genes, which we suggest in this study, did not match the results in elk [3]. The reason could not be explained, but there are some points which need to be considered. Firstly, the number of CWD affected elk in the study was limited to two. Secondly, the incubation period to the point of clinical symptom appearance was quite long (738 dpi) because the elk were inoculated orally with 1 g of brain tissue. Gene expression may be slower in elk as the disease progresses, compared with TgElk mice showing clinical signs at 185 dpi. This may explain the reason why the current result matches with those of other prion disease-affected mice models which do not overlap with those of large animals as in this study. Thirdly, the microarray analysis was based on the bovine genome due to a lack of information on the elk genome. It would be worthwhile to validate these suggested genes in an experimental infection which is in progress at the Canadian Food Inspection Agency (CFIA), Canada in the near future.  

Table 3. Comparison of gene expression profiles between elkRK13 versus RKC1-11 cells

Table 4. Oligonucleotides and probes used in qRT-PCR analysis

ACKNOWLEDGMENTS

We would like to thank the staff members of the Foreign Animal Disease Division, Animal Plant and Fisheries Quarantine and Inspection Agency. We are grateful to the New York State Institute for Basic Research in Developmental Disabilities, USA for supplying the transgenic mice over-expressing the elk prion protein. This work was funded by the Animal Plant and Fisheries Quarantine and Inspection Agency, the Republic of Korea (C-1541782-2012-14-01). 

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