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Transcriptional profiling of the effects of 25-hydroxycholesterol on human hepatocyte metabolism and the antiviral state it conveys against the hepatitis C virus.

Pezacki JP, Sagan SM, Tonary AM, Rouleau Y, Bélanger S, Supekova L, Su AI - BMC Chem Biol (2009)

Bottom Line: These data were compared with gene expression profiles from HCV-infected chimpanzees.Functional studies of 3 of these genes demonstrates that they do not directly act as antiviral gene products but that they indirectly contribute to the antiviral state in the host cell.These genes may also represent novel biomarkers for HCV infection, since they demonstrate an outcome-specific expression profile.

View Article: PubMed Central - HTML - PubMed

Affiliation: Steacie Institute for Molecular Sciences, The National Research Council of Canada, Ottawa, K1A 0R6 Canada . John.pezacki@nrc.ca

ABSTRACT

Background: Hepatitis C virus (HCV) infection is a global health problem. A number of studies have implicated a direct role of cellular lipid metabolism in the HCV life cycle and inhibitors of the mevalonate pathway have been demonstrated to result in an antiviral state within the host cell. Transcriptome profiling was conducted on Huh-7 human hepatoma cells bearing subgenomic HCV replicons with and without treatment with 25-hydroxycholesterol (25-HC), an inhibitor of the mevalonate pathway that alters lipid metabolism, to assess metabolic determinants of pro- and antiviral states within the host cell. These data were compared with gene expression profiles from HCV-infected chimpanzees.

Results: Transcriptome profiling of Huh-7 cells treated with 25-HC gave 47 downregulated genes, 16 of which are clearly related to the mevalonate pathway. Fewer genes were observed to be upregulated (22) in the presence of 25-HC and 5 genes were uniquely upregulated in the HCV replicon bearing cells. Comparison of these gene expression profiles with data collected during the initial rise in viremia in 4 previously characterized HCV-infected chimpanzees yielded 54 overlapping genes, 4 of which showed interesting differential regulation at the mRNA level in both systems. These genes are PROX1, INSIG-1, NK4, and UBD. The expression of these genes was perturbed with siRNAs and with overexpression vectors in HCV replicon cells, and the effect on HCV replication and translation was assessed. Both PROX1 and NK4 regulated HCV replication in conjunction with an antiviral state induced by 25-hydroxycholesterol.

Conclusion: Treatment of Huh-7 cells bearing HCV replicons with 25-HC leads to the downregulation of many key genes involved in the mevalonate pathway leading to an antiviral state within the host cell. Furthermore, dysregulation of a larger subset of genes not directly related to the mevalonate pathway occurs both in 25-HC-treated HCV replicon harbouring cells as well as during the initial rise in viremia in infected chimpanzees. Functional studies of 3 of these genes demonstrates that they do not directly act as antiviral gene products but that they indirectly contribute to the antiviral state in the host cell. These genes may also represent novel biomarkers for HCV infection, since they demonstrate an outcome-specific expression profile.

No MeSH data available.


Related in: MedlinePlus

The effect of 25-hydroxycholesterol and siRNA knockdown or gene overexpression on HCV RNA replication and translation. Huh-7 cells stably expressing neo/luc/NS3-3'/5.1 or neo/NS3-3'/5.1 subgenomic replicons were transiently transfected with siRNA duplexes homologous to the genes of interest (A and B) or with expression plasmids for the genes of interest (C and D) in the absence or presence of 5 μM 25-hydroxycholesterol (25-HC) for 48 hours. After siRNA knockdown or overexpression of target genes, cell lysates were analyzed for luciferase activity (A and C) and western blot analysis of HCV non-structural proteins NS3 and NS5A as well as PROX1 (B and D). GL2 and GL3 siRNAs were used as controls for siRNA knockdown as described in the Methods. Mock refers to cells that were incubated with the transfection reagent but without siRNA or plasmid. As a positive control, cells were separately treated with 100 U/ml IFNγ. Values in (A) and (C) are the mean normalized relative light units (RLU) per mg/ml of protein ± standard deviation from triplicate samples. For the siRNA knockdown (A), an asterisk above a bar indicates a significant decrease in HCV replication compared to mock-transfected cells (GL2, PROX-1; p < 0.01) or untreated cells (IFNγ; p < 0.01) in the presence of 5 μM 25-HC. For the overexpression studies (C), an asterisk above a bar indicates a significant decrease in HCV replication compared to cells transfected with the backbone β-gal vector (NK4; p < 0.05) or the untreated cells (IFNγ; p < 0.01) when compared to the appropriate treatment condition (no treatment or 5 μM 25-HC treatment as indicated).
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Figure 5: The effect of 25-hydroxycholesterol and siRNA knockdown or gene overexpression on HCV RNA replication and translation. Huh-7 cells stably expressing neo/luc/NS3-3'/5.1 or neo/NS3-3'/5.1 subgenomic replicons were transiently transfected with siRNA duplexes homologous to the genes of interest (A and B) or with expression plasmids for the genes of interest (C and D) in the absence or presence of 5 μM 25-hydroxycholesterol (25-HC) for 48 hours. After siRNA knockdown or overexpression of target genes, cell lysates were analyzed for luciferase activity (A and C) and western blot analysis of HCV non-structural proteins NS3 and NS5A as well as PROX1 (B and D). GL2 and GL3 siRNAs were used as controls for siRNA knockdown as described in the Methods. Mock refers to cells that were incubated with the transfection reagent but without siRNA or plasmid. As a positive control, cells were separately treated with 100 U/ml IFNγ. Values in (A) and (C) are the mean normalized relative light units (RLU) per mg/ml of protein ± standard deviation from triplicate samples. For the siRNA knockdown (A), an asterisk above a bar indicates a significant decrease in HCV replication compared to mock-transfected cells (GL2, PROX-1; p < 0.01) or untreated cells (IFNγ; p < 0.01) in the presence of 5 μM 25-HC. For the overexpression studies (C), an asterisk above a bar indicates a significant decrease in HCV replication compared to cells transfected with the backbone β-gal vector (NK4; p < 0.05) or the untreated cells (IFNγ; p < 0.01) when compared to the appropriate treatment condition (no treatment or 5 μM 25-HC treatment as indicated).

Mentions: To ascertain the importance of the four genes for HCV replication, siRNA knockdown and overexpression studies were carried out in Huh-7 cells harbouring HCV replicon RNA. RT-PCR and northern blot analyses were used to confirm siRNA and overexpression vector function in each case (data not shown). Huh-7 cells stably expressing neo/luc/NS3-3'/5.1 HCV replicons [8,25-27] were treated with siRNAs against the four genes in the absence or presence of 25-HC (Fig. 5A). As controls, the HCV replicon cells were also treated with GL2 and GL3 siRNAs, a positive control that targets the luciferase gene expressed from the HCV replicon RNA and a negative control, respectively, in addition to IFNγ treatment [15]. Interestingly, although the PROX1 siRNA did not affect HCV replication on its own, it enhanced the inhibitory effect of treatment with 5 μM 25-HC when compared to mock-transfected replicon cells treated with 5 μM 25-HC (p < 0.01) as indicated (Fig. 5A). Overexpression of PROX1 in replicon cells did not affect HCV replication (Fig. 5C). Knockdown of INSIG-1 with homologous siRNAs did not affect HCV replication in replicon cells either in the absence or presence of 25-HC (Fig. 5A). Similarly, overexpression of INSIG-1 in replicon cells did not affect HCV replication (Fig. 5C). Although the transcriptional profiling showed that NK4 was upregulated in replicon cells treated with 25-HC, siRNA knockdown of NK4 did not affect HCV replication (Fig. 5A). However, overexpression of NK4 affected HCV replication in the presence of 25-HC treatment, causing a significant decrease in HCV replication compared to replicon cells that were transfected with the backbone β-gal control vector and treated with 25-HC (p < 0.05; Fig. 5C). From the profiling data, UBD was upregulated in 25-HC-treated replicon cells compared with naïve Huh-7 cells treated with 25-HC. However, neither knockdown of UBD using siRNA duplexes (Fig. 5A) nor overexpression of UBD (Fig. 5C) had an affect on HCV replication.


Transcriptional profiling of the effects of 25-hydroxycholesterol on human hepatocyte metabolism and the antiviral state it conveys against the hepatitis C virus.

Pezacki JP, Sagan SM, Tonary AM, Rouleau Y, Bélanger S, Supekova L, Su AI - BMC Chem Biol (2009)

The effect of 25-hydroxycholesterol and siRNA knockdown or gene overexpression on HCV RNA replication and translation. Huh-7 cells stably expressing neo/luc/NS3-3'/5.1 or neo/NS3-3'/5.1 subgenomic replicons were transiently transfected with siRNA duplexes homologous to the genes of interest (A and B) or with expression plasmids for the genes of interest (C and D) in the absence or presence of 5 μM 25-hydroxycholesterol (25-HC) for 48 hours. After siRNA knockdown or overexpression of target genes, cell lysates were analyzed for luciferase activity (A and C) and western blot analysis of HCV non-structural proteins NS3 and NS5A as well as PROX1 (B and D). GL2 and GL3 siRNAs were used as controls for siRNA knockdown as described in the Methods. Mock refers to cells that were incubated with the transfection reagent but without siRNA or plasmid. As a positive control, cells were separately treated with 100 U/ml IFNγ. Values in (A) and (C) are the mean normalized relative light units (RLU) per mg/ml of protein ± standard deviation from triplicate samples. For the siRNA knockdown (A), an asterisk above a bar indicates a significant decrease in HCV replication compared to mock-transfected cells (GL2, PROX-1; p < 0.01) or untreated cells (IFNγ; p < 0.01) in the presence of 5 μM 25-HC. For the overexpression studies (C), an asterisk above a bar indicates a significant decrease in HCV replication compared to cells transfected with the backbone β-gal vector (NK4; p < 0.05) or the untreated cells (IFNγ; p < 0.01) when compared to the appropriate treatment condition (no treatment or 5 μM 25-HC treatment as indicated).
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Figure 5: The effect of 25-hydroxycholesterol and siRNA knockdown or gene overexpression on HCV RNA replication and translation. Huh-7 cells stably expressing neo/luc/NS3-3'/5.1 or neo/NS3-3'/5.1 subgenomic replicons were transiently transfected with siRNA duplexes homologous to the genes of interest (A and B) or with expression plasmids for the genes of interest (C and D) in the absence or presence of 5 μM 25-hydroxycholesterol (25-HC) for 48 hours. After siRNA knockdown or overexpression of target genes, cell lysates were analyzed for luciferase activity (A and C) and western blot analysis of HCV non-structural proteins NS3 and NS5A as well as PROX1 (B and D). GL2 and GL3 siRNAs were used as controls for siRNA knockdown as described in the Methods. Mock refers to cells that were incubated with the transfection reagent but without siRNA or plasmid. As a positive control, cells were separately treated with 100 U/ml IFNγ. Values in (A) and (C) are the mean normalized relative light units (RLU) per mg/ml of protein ± standard deviation from triplicate samples. For the siRNA knockdown (A), an asterisk above a bar indicates a significant decrease in HCV replication compared to mock-transfected cells (GL2, PROX-1; p < 0.01) or untreated cells (IFNγ; p < 0.01) in the presence of 5 μM 25-HC. For the overexpression studies (C), an asterisk above a bar indicates a significant decrease in HCV replication compared to cells transfected with the backbone β-gal vector (NK4; p < 0.05) or the untreated cells (IFNγ; p < 0.01) when compared to the appropriate treatment condition (no treatment or 5 μM 25-HC treatment as indicated).
Mentions: To ascertain the importance of the four genes for HCV replication, siRNA knockdown and overexpression studies were carried out in Huh-7 cells harbouring HCV replicon RNA. RT-PCR and northern blot analyses were used to confirm siRNA and overexpression vector function in each case (data not shown). Huh-7 cells stably expressing neo/luc/NS3-3'/5.1 HCV replicons [8,25-27] were treated with siRNAs against the four genes in the absence or presence of 25-HC (Fig. 5A). As controls, the HCV replicon cells were also treated with GL2 and GL3 siRNAs, a positive control that targets the luciferase gene expressed from the HCV replicon RNA and a negative control, respectively, in addition to IFNγ treatment [15]. Interestingly, although the PROX1 siRNA did not affect HCV replication on its own, it enhanced the inhibitory effect of treatment with 5 μM 25-HC when compared to mock-transfected replicon cells treated with 5 μM 25-HC (p < 0.01) as indicated (Fig. 5A). Overexpression of PROX1 in replicon cells did not affect HCV replication (Fig. 5C). Knockdown of INSIG-1 with homologous siRNAs did not affect HCV replication in replicon cells either in the absence or presence of 25-HC (Fig. 5A). Similarly, overexpression of INSIG-1 in replicon cells did not affect HCV replication (Fig. 5C). Although the transcriptional profiling showed that NK4 was upregulated in replicon cells treated with 25-HC, siRNA knockdown of NK4 did not affect HCV replication (Fig. 5A). However, overexpression of NK4 affected HCV replication in the presence of 25-HC treatment, causing a significant decrease in HCV replication compared to replicon cells that were transfected with the backbone β-gal control vector and treated with 25-HC (p < 0.05; Fig. 5C). From the profiling data, UBD was upregulated in 25-HC-treated replicon cells compared with naïve Huh-7 cells treated with 25-HC. However, neither knockdown of UBD using siRNA duplexes (Fig. 5A) nor overexpression of UBD (Fig. 5C) had an affect on HCV replication.

Bottom Line: These data were compared with gene expression profiles from HCV-infected chimpanzees.Functional studies of 3 of these genes demonstrates that they do not directly act as antiviral gene products but that they indirectly contribute to the antiviral state in the host cell.These genes may also represent novel biomarkers for HCV infection, since they demonstrate an outcome-specific expression profile.

View Article: PubMed Central - HTML - PubMed

Affiliation: Steacie Institute for Molecular Sciences, The National Research Council of Canada, Ottawa, K1A 0R6 Canada . John.pezacki@nrc.ca

ABSTRACT

Background: Hepatitis C virus (HCV) infection is a global health problem. A number of studies have implicated a direct role of cellular lipid metabolism in the HCV life cycle and inhibitors of the mevalonate pathway have been demonstrated to result in an antiviral state within the host cell. Transcriptome profiling was conducted on Huh-7 human hepatoma cells bearing subgenomic HCV replicons with and without treatment with 25-hydroxycholesterol (25-HC), an inhibitor of the mevalonate pathway that alters lipid metabolism, to assess metabolic determinants of pro- and antiviral states within the host cell. These data were compared with gene expression profiles from HCV-infected chimpanzees.

Results: Transcriptome profiling of Huh-7 cells treated with 25-HC gave 47 downregulated genes, 16 of which are clearly related to the mevalonate pathway. Fewer genes were observed to be upregulated (22) in the presence of 25-HC and 5 genes were uniquely upregulated in the HCV replicon bearing cells. Comparison of these gene expression profiles with data collected during the initial rise in viremia in 4 previously characterized HCV-infected chimpanzees yielded 54 overlapping genes, 4 of which showed interesting differential regulation at the mRNA level in both systems. These genes are PROX1, INSIG-1, NK4, and UBD. The expression of these genes was perturbed with siRNAs and with overexpression vectors in HCV replicon cells, and the effect on HCV replication and translation was assessed. Both PROX1 and NK4 regulated HCV replication in conjunction with an antiviral state induced by 25-hydroxycholesterol.

Conclusion: Treatment of Huh-7 cells bearing HCV replicons with 25-HC leads to the downregulation of many key genes involved in the mevalonate pathway leading to an antiviral state within the host cell. Furthermore, dysregulation of a larger subset of genes not directly related to the mevalonate pathway occurs both in 25-HC-treated HCV replicon harbouring cells as well as during the initial rise in viremia in infected chimpanzees. Functional studies of 3 of these genes demonstrates that they do not directly act as antiviral gene products but that they indirectly contribute to the antiviral state in the host cell. These genes may also represent novel biomarkers for HCV infection, since they demonstrate an outcome-specific expression profile.

No MeSH data available.


Related in: MedlinePlus