HCV Tumor Promoting Effect Is Dependent on Host Genetic Background

Background The hepatitis C virus (HCV) is one of the major risk factors for the development of hepatocellular carcinoma (HCC). Nevertheless, transgenic mice which express the whole HCV polyprotein (HCV-Tg) do not develop HCC. Whereas chronic HCV infection causes inflammation in patients, in HCV-Tg mice, the host immune reaction against viral proteins is lacking. We aimed to test the role of HCV proteins in HCC development on the background of chronic inflammation in vivo. Methodology/Principal Findings We crossed HCV-Tg mice that do not develop HCC with the Mdr2-knockout (Mdr2-KO) mice which develop inflammation-associated HCC, to generate Mdr2-KO/HCV-Tg mice. We studied the effect of the HCV transgene on tumor incidence, hepatocyte mitosis and apoptosis, and investigated the potential contributing factors for the generated phenotype by gene expression and protein analyses. The Mdr2-KO/HCV-Tg females from the N2 generation of this breeding (having 75% of the FVB/N genome and 25% of the C57BL/6 genome) produced significantly larger tumors in comparison with Mdr2-KO mice. In parallel, the Mdr2-KO/HCV-Tg females had an enhanced inflammatory gene expression signature. However, in the N7 generation (having 99.2% of the FVB/N genome and 0.8% of the C57BL/6 genome) there was no difference in tumor development between Mdr2-KO/HCV-Tg and Mdr2-KO animals of both sexes. The HCV transgene was similarly expressed in the livers of Mdr2-KO/HCV-Tg females of both generations, as revealed by detection of the HCV transcript and the core protein. Conclusion These findings suggest that the HCV transgene accelerated inflammation-associated hepatocarcinogenesis in a host genetic background-dependent manner.


Introduction
Hepatocellular carcinoma (HCC) is a significant health care burden worldwide, and chronic inflammation is a major risk factor for the development of HCC. The hepatitis C virus (HCV) is one of the major etiological HCC agents: it induces chronic liver inflammation and is responsible for the increased incidence of HCC in the developed world [1]. Although multiple cell culture models demonstrated oncogenic potential of several HCV proteins, most transgenic mouse models expressing these proteins did not develop HCC [2]. Furthermore, non infectious metabolic conditions causing chronic hepatitis also expose humans to an increased risk of developing HCC. Thus, determinants of HCC development on the background of chronic inflammation could be attributed to genetic/gender background, pathogen-derived or environmentally-dependent factors [3][4][5]. The aim of this study is to investigate the interplay between the factors contributing to the development of HCC in a mouse model expressing HCV proteins.
The previously generated HCV transgenic (HCV-Tg) mouse models produced inconsistent results concerning the role of HCV proteins in HCC development. Only one model that expressed the HCV core protein developed HCC with high incidence [6], and two models that expressed the HCV core together with E1 and E2 proteins developed HCC with very low incidence [7,8]. However, other models containing similar transgenes, albeit produced in other laboratories, did not develop HCC [2]. Two groups created HCV-Tg mice expressing the whole HCV polyprotein [7,9], however, none of these models are developing HCC (although in one model, HCC development was reported [7], this phenotype disappeared in the progeny throughout the next generations [10]). In transgenic animals, there is no inflammatory reaction against proteins expressed by a transgene, whereas in HCV-infected patients, the liver is inflamed. On the other hand, genetic models with chronic liver inflammation develop HCC at a high rate [11][12][13]. We and others have reported the importance of inflammatory mediators in HCC development [14][15][16][17].
In order to explore the hypothesis that HCV proteins may accelerate inflammation-associated liver carcinogenesis, and to identify and characterize the signaling pathways most likely to be affected, we crossed one of the existing HCV-Tg models, which produced detectable levels of HCV proteins [9], with the Mdr2 knockout (Mdr2-KO) mice, a model of inflammation-associated HCC. The Mdr2-KO mice develop liver tumors in about 100% of animals by the age of 16 months on the FVB/N genetic background; initially, this model was generated in the 129/ OlaHsd strain, but due to a low fertility of the resulting mutants, the Mdr2-KO mutation was transferred into the FVB/N strain [11,18]. We monitored HCC development in these hybrid mice by ultrasound, and, upon sacrifice, analyzed mitotic, apoptotic and gene expression patterns in their tumorous and non-tumorous liver tissues.

Mice
Mice were maintained at the Specific Pathogen-Free (SPF) unit, Faculty of Medicine, Hebrew University, under a 12 h light/dark cycle, and provided with food and water ad libitum. All animal experiments were performed according to national regulations and guidelines of the Institutional Animal Welfare Committee (NIH approval number OPRR-A01-5011). Founders of the FVB.129P2-Abcb4 tm1Bor (Mdr2-KO; old name FVB.129P2-Pgy24 tm1Bor ) mice were purchased from the Jackson Laboratory (Bar Harbor, USA) [11]. Transgenic mice expressing the whole HCV 1b polyprotein were kindly provided by Prof. N. La Monica (IRBM, ''P. Angeletti'', Pomezia, Italy) [9]. Double Mdr2-KO/HCV-Tg mice were produced by crossing between homozygous Mdr2-KO and HCV-Tg mice, and by backcrosses of the resulting F1 hybrid (both Mdr2-and HCV-heterozygous) with the parental Mdr2-KO strain. Appearance of tumors was monitored by ultrasound from the age of 10 months, using an ATL 5000 device (ATL, Bothell, WA) with linear transducer (12)(13)(14)(15). Mice were sacrificed at the age of 14 months, when tumor appearance was detected in most Mdr2-KO mice.
Genotyping DNA from mouse tails or livers was purified by either the WizardH Genomic DNA purification kit (Promega, Madison, WI) or by the PuregeneH DNA isolation kit (Gentra systems, Minneapolis, MN). Genotype analysis was carried out by PCR analysis of DNA extracted from mouse tails using HCV transgene, Mdr2 and Neo primers (primers sequences are specified in the Table 1).

Harvesting of mouse tissue
Non fasting mice were anesthetized with isoflurane and sacrificed by cervical dislocation. Livers were rapidly excised and weighed; a part of the liver was fixed with 4% buffered formaldehyde for histological analysis, and the remaining tissue was rapidly frozen in liquid nitrogen and stored at 280uC until further use.

RNA extraction and analysis
Total RNA was isolated from snap frozen mouse liver tissues with the TrizolH reagent (Invitrogen, Carlsbad, CA) as described by the manufacturer. Whole genome gene expression profiling of RNA samples was performed using Affymetrix GeneChipH Mouse Exon 1.0 ST Array (Affymetrix, Santa Clara, CA).

Calculation of gene expression values
The raw data were processed by the PartekH Genomics Suite TM (Partek GS) (Partek Inc., St. Louis, MO) software. RMA algorithm was applied for summarization, and the normalization was performed by the quantile method. The normalized expression values were processed by the Partek batch removing tool. The analysis was repeated twice: on ''extended'' and ''core'' subsets of probe-sets and when the probe-set mean was applied for evaluation of transcript cluster expression level. The processed data were submitted either to fold-change analysis with a threshold of 1.85, to hierarchical clustering with the Eisen software [19], or to SPIN analysis (sorting points into neighborhoods (sorting algorithm)) [20], which enables arrangement of expression profiles in the order of similarity (''side-to-side'' sorting variant).

Analysis of alternative splicing
The alternative splicing calculation was performed using the definition of splicing index as average log 2 transformed relations between expression levels of individual probe-sets (representing individual exons) and transcript clusters (evaluation of gene expression level) [21]. Statistical significance of the differences between splicing indices in different experimental groups was evaluated with the ANOVA test for comparison between the nonmatched samples or paired T-test for matched samples (tumors and non-tumorous tissues). The significant difference in the splicing index was considered to be a sign of change of the tested exon inclusion levels. Only probe-sets and transcripts with a maximal expression level .5 (in log 2 scale) were used.

Confirmation of differential gene expression by RT-PCR
Reverse transcription of total RNA was performed using the MMLV Reverse Transcriptase and random hexamer primers (Promega, Madison, WI). Semi-quantitative PCR was performed using Super-therm Taq Polymerase (JMR, UK). For each gene,

Statistical analysis of the data
Significance of the difference in tumor incidence between mouse groups was tested by Chi-Square-test; significance of differences in the levels of blood enzymes, in the levels galectin-1 immunostaining, in the frequency of mitosis and apoptosis, and in the semi-quantitative RT-PCR was tested by t-test. Statistical evaluation of differential expression between the experimental groups was performed using ANOVA test.

Effect of the HCV transgene on hepatocarcinogenesis in Mdr2-KO mice
The first breeding between homozygous HCV-Tg and Mdr2-KO mice (having C57Bl/6 and FVB/N genetic backgrounds, respectively) produced a generation of HCV-and Mdr2heterozygotes (Fig. S1). The second backcross of these mice to the maternal Mdr2-KO strain produced the N2 generation which contained 25% of the C57Bl/6 genetic background. We continued to produce backcrosses till the N7 generation, an incipient congenic strain containing only 0.8% of the donor C57Bl/6 genetic background and 99.2% of the recipient FVB/N genetic background. The produced hybrid mice were followed for HCC development, and comparison between the N2 and N7 generations was performed. Independently of the presence of the HCV transgene, Mdr2 +/2 mice did not develop HCC upon sacrifice at 18 months of age, whereas all Mdr2-KO mice did produce liver tumors at 14 months of age (Table 2). Most tumors were welldifferentiated HCC as determined by previously described criteria [18] and by immunostaining for reticulin fibers (not shown). Immunohistochemical analysis of liver tissues from HCV-Tg mice with antibodies to the core protein showed non-uniform distribution of HCV transgene expression both in Mdr2-KO and Mdr2-heterozygous mice. Levels of the HCV transgene expression were similar between Mdr2-KO/HCV-Tg mice from the N2 and N7 generations (Fig. 1, A and B). However, a significant difference in the incidence of large tumors was observed upon comparing N2 female mice with and without the HCV transgene: only N2 female mice with the HCV transgene had tumors with diameters over 1 cm (p = 0.0445; Table 2 and Fig. 1C). In contrast, in mice from the N7 generation, the incidence of large tumors was independent of the presence of the HCV transgene and was similar to that in Mdr2-KO/HCV-Tg mice from the N2 generation. The 25% of the C57Bl/6 genetic background present in the N2 generation had a tumor suppressive effect in the Mdr2-KO mice not having the HCV transgene (Fig. 1C). There was a statistically significant reduced incidence of tumors with a diameter over 1 cm between the animals from the N2 and N7 generations (Chi-Square-tests for both all mice (p = 0.0237) and for females (p = 0.0299); Table 2). The tumor promoting effect of the HCV transgene was statistically significant only on the partially suppressive N2 genetic background, and only in females (p = 0.0445).
Higher levels of liver enzymes (alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase) were found in the serum of all Mdr2-KO mice compared to Mdr2 +/2 controls ( Fig. 2A), as expected [15]. However, the only statistically significant difference between Mdr2-KO/HCV mice and Mdr2-KO mice from the N2 generation was in the level of alkaline phosphatase, found in females only (p = 0.04). Nevertheless, there was no direct correlation between the level of alkaline phosphatase and liver tumor load/incidence in individual Mdr2-KO/HCV mice. Similarly, high levels of serum liver enzymes were also measured in Mdr2-KO mice of the N7 generation; however, the level of alkaline phosphatase was not specifically increased in the Mdr2-KO/HCV-Tg females compared to Mdr2-KO females not expressing the HCV transgene (Fig. 2B).

Effect of the HCV transgene on mitosis and apoptosis in Mdr2-KO mice
To investigate the potential mechanisms for the effect of the HCV transgene on hepatocarcinogenesis, we compared mitotic and apoptotic events in non-tumorous and tumorous liver tissues of the Mdr2-KO and Mdr2-KO/HCV-Tg females from the N2 and N7 generations ( Fig. 3A -mitosis, Fig. 3B -apoptosis) and of the males from the N2 generation (Fig. 3C). Interestingly, we observed a statistically significant decrease of mitosis (p,0.016) and apoptosis (p,0.002) in the non-tumorous tissues of the Mdr2-KO/HCV-Tg females from the N2 generation only; however, once the anti-mitotic effect of the HCV transgene was bridged in tumors, then mitosis was enhanced in both female and male mice from the N2 generation ( Fig. 3A and Fig. 3C). This effect was more prominent in female mice, although it did not reach statistical significance. The presence of the highly proliferative  tumors with diameters of up to 1 cm in Mdr2-KO/HCV-Tg females indicates a possible reason for the appearance of large tumors with diameters over 1 cm in this experimental group (Fig. 1C) as well. In the N7 females, the anti-mitotic effect of the HCV transgene on non-tumorous liver tissue was absent, and the mitotic activities of tumors in the Mdr2-KO and Mdr2-KO/ HCV-Tg females were similar to each other and to those of the Mdr2-KO females from the N2 generation (Fig. 3A). The levels of apoptosis in tumors were similar between N2 and N7 females, independent of the presence of the HCV transgene (Fig. 3B).

Gene expression profiling analysis
In an effort to delineate the molecular mechanisms associated with the tumor promoting effect of the HCV transgene in Mdr2-KO/HCV-Tg N2 females, we performed a genome scale gene expression profiling on livers of 14-month-old female mice. Nontumorous liver tissues of Mdr2-KO, Mdr2 +/2 , Mdr2 +/2 /HCV-Tg, and Mdr2-KO/HCV-Tg female mice from the N2 generation, as well as all large tumors (diameters over 1 cm) of Mdr2-KO/HCV-Tg females, were analyzed (Fig. 4). In hierarchical clustering, the Mdr2 genotype and the tumor phenotype were major clustering forces separating the samples into three main clusters: Mdr2-KO, Mdr2 +/2 and tumors. Remarkably, in the cluster of healthy Mdr2 +/2 mice, the HCV transgene was a destabilizing factor, whereas in the chronically inflamed Mdr2-KO background, it was a stabilizing factor providing clusterization of all HCV-Tg samples into one subcluster. Similarly, Mdr2-KO mutation was the main factor contributing to the alternative splicing between the experimental groups (Table S1). Overall, the contribution of the HCV transgene in both differential gene expression (Fig. S2) and alternative splicing (Table S1) was only marginal. However, supervised analysis of genes with a tendency to differential expression between Mdr2-KO and Mdr2-KO/ HCV-Tg samples revealed a substantial overlap of pathways affected by the HCV transgene and the Mdr2-KO mutation (Table S2). This overlap is comprised of all arms of the immune system, including the innate, humoral and, to some extent, the adaptive responses; many of these genes were also similarly differentially expressed between Mdr2 +/2 and Mdr2 +/2 /HCV-Tg samples (data not shown).
Up-regulation of two proteins, galectin-1 (encoded by the Lgals1 gene) and diubiquitin (encoded by the Ubd/Fat10 gene), was confirmed by immunohistochemistry. Galectin-1 modulates innate and adaptive immune responses; its over-expression in many types of tumors and/or surrounding tissues promotes tumor progression by inhibition of anti-tumor immune response [26]. Galectin-1 was overexpressed in non-tumorous liver tissue of Mdr2-KO/HCV-Tg mice, mostly in the cytoplasm of hepatocytes in zones 1 and 3, in endothelial cells, and on the luminal side of cholangiocyte membranes (Fig. 5). Diubiquitin is involved in the maintenance of spindle integrity during mitosis and is highly upregulated in human HCC [27]; it was detected mostly in the cytoplasm of hepatocytes in the tumors of Mdr2-KO/HCV-Tg mice, and, to a lesser degree, in non-tumorous liver tissues of Mdr2-KO mice with or without the HCV transgene (Fig. 6). Remarkably, diubiquitin was detected in the hepatocyte nuclei only in large tumors of Mdr2-KO/HCV-Tg mice, similar to its nuclear localization in human HCC [27].

Testing of other known effects of the HCV transgene expression
We searched for a correlation between the local level of the HCV transgene expression (revealed by immunohistochemical detection of the HCV core protein) and other oncogenic markers. There was no correlation between the level of HCV transgene expression and the presence of dysplastic nodules in Mdr2-KO/ HCV-Tg mice. It was shown previously that HCV-associated HCC is characterized by increased frequency of b-catenin mutations and by activation of b-catenin signaling [28,29]. We reported recently that in the non-tumorous liver of Mdr2-KO mice, b-catenin signaling is inhibited while the nuclear level of cyclin D1 is increased [18]. The expression level of the HCV transgene in hepatocytes of Mdr2-KO/HCV-Tg mice correlated neither with the nuclear level of cyclin D1 nor with the cytoplasmic/nuclear level of b-catenin (a typical example shown in Fig. 7A). However, in some rare cases, high HCV transgene expression correlated with nuclear localization of b-catenin (Fig. 7B). One of the six tumors analyzed by whole genome gene expression profiling was characterized by a prominent upregulation of the b-catenin-activated genes and by rare scattered bcatenin-positive hepatocyte nuclei (data not shown).

Discussion
The molecular mechanisms of HCV-induced HCC development are still not well understood; one of the main questions is whether HCC develops due to chronic inflammation or whether HCV proteins are endowed with some oncogenic activity.
Multiple cell culture models have demonstrated the oncogenic potential of several HCV proteins; however, most transgenic mouse models expressing HCV proteins did not develop HCC [2]. Similarly, none of the two previously generated transgenic mouse models expressing the whole HCV polyprotein is developing HCC [7,9,10]. Taking into account that in mouse transgenic models there is no host immune reaction against expressed viral proteins, we explored whether HCV proteins would accelerate inflammation-associated hepatocarcinogenesis. We bred mice expressing the whole HCV polyprotein with Mdr2-KO mice, a model of inflammation-associated HCC, and monitored HCC development in two generations of hybrid mice: N2 and N7, containing 25% and 0.8% of C57Bl/6 genetic background, respectively. We demonstrated, for the first time, that a transgene expressing the whole HCV polyprotein accelerates inflammation-associated hepatocarcinogenesis in vivo in a host genetic backgrounddependent manner: only in the N2 generation and only in females. Remarkably, our previous studies of Mdr2-KO mice [15] demonstrated that chronic liver disease at an early precancerous stage in this HCC model shares many features characteristic of chronic HCV infection and HCV-induced cirrhosis in patients: hepatocyte G1 arrest and up-regulation of genes Mcm2 [30], Cxcl9, Lgals3, Iqgap1, Lum, Col6a3, Igfbp7, Vim, Ablim, Mpp1  [31], Igfbp1, Itgb1, Jun, Ctgf [32]. This similarity was further confirmed by a significant overlap of pathways affected by the HCV transgene and the Mdr2-KO mutation revealed in the current study (Table S2).
The 25% of C57Bl/6 genetic background had a tumorsuppressive effect on HCC development in the N2 mice not having the HCV transgene, in accord with a well known low susceptibility of the C57Bl/6 mice to tumors [33]. Thus, in the PyMT mouse model of breast cancer, C57Bl/6 mice and F1 hybrids between the C57Bl/6 and FVB/N strains had more prolonged primary tumor latency than the FVB/N mice [34,35]. Remarkably, the effect of inducible nitric oxide synthase deficiency on mammary tumor development and metastasis was prominent in the C57Bl/6 genetic background, where tumors developed slowly, but was not observed in the FVB/N genetic background, where tumors developed quickly [34]. In the Ras-induced skin squamous carcinoma model, the low susceptibility of the C57Bl/6 strain and the high susceptibility of the FVB/N had been attributed to a polymorphism in the Ptch gene [36]. Interestingly, it was recently demonstrated that inactivation of the Nox2 gene in the mutant Sod1 mouse model for amyotrophic lateral sclerosis significantly increased survival when mice were of mixed genetic background [37], but had very modest effect on the inbred C57Bl/ 6 strain [38]. In accord with these findings, our data demonstrate that for mouse models of human diseases, a specific genetic Table 3. Confirmation of the differential gene expression by RT-PCR.  [27], resulting in chromo-some instability [60].
Vnn1 @ NM_011704 T.NT p = 0.01 vanin 1 Regulates response to oxidative injury in epithelial cells [61].  background may be found where manifestations of the disease and/or therapeutic treatments will be the most prominent.
The female-specific effect of the HCV transgene raises a question about the relevance of this model to human disease, since it is widely accepted that human HCC is more prevalent in males. The typical average ratio of males:females among HCC patients is 3:1 [39], however, this proportion depends on the etiology of the disease: among HBV-infected patients, it is about 6:1, whereas among HCV-infected patients, it is about 1.7:1 [40]. Moreover, when other higher HCC risk factors in males such as alcohol consumption are taken into account, the normalized risk of HCC among HCV-infected patients may be higher for females [41].
Therefore, the appearance of the tumor-promoting effect of the HCV transgene seen only in females in the described model may have a physiological significance. Recently, it was demonstrated that in chemically induced hepatocarcinogenesis in C57Bl/6 mice, the estrogen-mediated inhibition of IL-6 production is responsible for a lower risk of liver cancer in females [3]. However, in the Mdr2-KO mice, the risk for liver cancer is similar in both genders [11,42], suggesting that there are probably other mechanisms regulating hepatocarcinogenesis in this model of chronic inflammation-associated HCC.
Comparison of mitotic and apoptotic events in non-tumorous liver tissues of the Mdr2-KO and Mdr2-KO/HCV-Tg mice revealed a statistically significant decrease of mitosis and apoptosis in the Mdr2-KO/HCV-Tg females from the N2 generation. Interestingly, similar effects of the transgene expressing the HCV open reading frame on the untransformed human hepatocytes have been demonstrated recently: absence of the direct oncogenic activity, inhibition of cell proliferation, and up-regulation of genes involved in innate immune response/inflammation [43]. However, in tumors, an inverse tendency took place: Mdr2-KO/HCV-Tg mice had higher levels of both mitosis and apoptosis, whereas in Mdr2-KO mice, the levels of mitosis and apoptosis in nontumorous and tumorous tissues were similar. These findings suggest that the accelerating effect of the HCV transgene on hepatocarcinogenesis in this model is most probably due to promotion of tumor proliferation by the HCV transgene. Similarly, the difference in liver cancer susceptibility between the C3H/He and C57Bl/6 strains has been attributed to a dissimilar tumor cell proliferation between these two groups, but not to intensity of apoptosis in their livers [44]. High standard errors observed in the current study for many parameters at the tumorous stage of the disease (e.g., hepatocyte's apoptotic and mitotic frequencies) were most probably caused by a prolonged  The tumor promoting effect of the HCV transgene may be explained both by upregulation of pro-tumorigenic genes as Ubd, Erbb4, Nrg1, Ndrg1, Styk1 (Table 3), and by evasion of host defense response by viral proteins [45,46]. Over-expression of the galectin-1 protein in the liver of Mdr2-KO/HCV-Tg mice may also cause inhibition of anti-tumor immune response [26]. One of the known differences between the C57Bl/6 and FVB/N inbred strains is that the latter is C5 complement-deficient. The important role of C5 and its receptor C5a in host inflammatory reactions [47] suggests that this factor may be responsible for the observed differences in HCC development between the N2 and N7 generations. Interestingly, comparison of differentially expressed genes (Mdr2-KO relative to control mice) of the N2 generation and of FVB/N Mdr2-KO mice aged 12 months [15] or 16 months [18], previously published by our group, revealed significant up-regulation of the complement component C9 in the Mdr2-KO livers of the N2 generation (4fold; p = 0.0066), but not in FVB/N Mdr2-KO mice. And conversely, inflammatory markers Aif1, Cd36, Cd44 and Spp1 (osteopontin) were up-regulated in FVB/N Mdr2-KO mice at all tested ages, but not in the Mdr2-KO livers of the N2 generation.
This study demonstrates that the HCV transgene accelerated inflammation-associated hepatocarcinogenesis in vivo in a host genetic background-dependent manner, thus emphasizing the significance of the host genotype in HCV manifestations. This accelerating effect was prominent in the mixed, partially tumorsuppressive, FVB/N6C57Bl/6 genetic background, and is most probably due to promotion of tumor proliferation by the HCV transgene. Despite multiple reports on the direct oncogenic activities of different HCV proteins in vitro, in mouse models in vivo, these data are contradictory, whereas data on the tumor promoting effect of HCV proteins are much more consistent [10,23,48]. Our data suggest that the selected Mdr2-KO mouse model was appropriate for this specific investigation aimed to determine which of the potential different factors contributed mainly to HCC development with special emphasis on the inflammatory process in the Mdr2-KO mice lacking the HCV transgene as its main contribution to the model. The role of specific factors contributing to tumor promoting activity of the HCV transgene: sex, known genes that differ between the two studied mouse strains, and differentially expressed regulatory genes revealed by our gene expression profiling analysis requires further investigation. Table S1

Supporting Information
The effect of Mdr2-KO, HCV-Tg, and tumor phenotype on alternative splicing. Numbers before slash represent the amount of probe-sets with 2-fold or 1.8-fold change in the relative level of exon inclusion, determined as statistically significant by either paired t-test (for tumors versus non-tumorous tissues), or two-way ANOVA (for all other categories). Numbers after slash represent the amount of unique genes represented by the selected probe-sets. * The data for alternative splicing in tumors could not be directly compared with other categories due to the use of a different method of calculation. Found at: doi:10.1371/journal.pone.0005025.s003 (0.03 MB DOC) Table S2 Substantial overlapping of pathways affected by the HCV transgene and by the Mdr2-KO mutation. The SPIN analysis of the complete set of samples performed in the space of the 1,000 most variable genes resulted in separation of the samples into 3 groups: non-tumorous Mdr2-KO, Mdr2(+/2), and tumorous Mdr2-KO/HCV-Tg samples (Fig. S2, left). The nontumorous Mdr2-KO set was roughly separated into Mdr2-KO/ HCV-Tg and Mdr2-KO subsets. The SPIN analysis of the selected gene-set in the space of all Mdr2-KO samples enabled isolation of genes with tendency to differential expression between HCV-Tg and non-HCV samples. The lists of genes with tendency to up-and down-regulation in Mdr2-KO/HCV-Tg samples were submitted to functional analysis by GO categories using the DAVID tool. These results were compared to the results of functional analysis by GO categories of genes differentially expressed between Mdr2-KO and Mdr2(+/2) non-HCV samples (threshold 1.85). For each enriched GO term, the number of related genes, their percent in the analyzed gene list and the significance of the enrichment are shown. Found at: doi:10.1371/journal.pone.0005025.s004 (0.03 MB DOC)