The multifactorial and multistage pathogenesis of hepatocellular carcinoma(HCC)has fascinated a wide spectrum of scientists for decades.While a number of major risk factors have been identified,their mechanistic roles...The multifactorial and multistage pathogenesis of hepatocellular carcinoma(HCC)has fascinated a wide spectrum of scientists for decades.While a number of major risk factors have been identified,their mechanistic roles in hepatocarcinogenesis still need to be elucidated.Many tumor suppressor genes(TSGs)have been identified as being involved in HCC.These TSGs can be classified into two groups depending on the situation with respect to allelic mutation/loss in the tumors:the recessive TSGs with two required mutated alleles and the haploinsufficient TSGs with one required mutated allele.Hepatitis B virus(HBV)is one of the most important risk factors associated with HCC.Although mice cannot be infected with HBV due to the narrow host range of HBV and the lack of a proper receptor,one advantage of mouse models for HBV/HCC research is the numerous and powerfulgenetic tools that help investigate the phenotypic effects of viral proteins and allow the dissection of the dose-dependent action of TSGs.Here,we mainly focus on the application of mouse models in relation to HBV-associated HCC and on TSGs that act either in a recessive or in a haploinsufficient manner.Discoveries obtained using mouse models will have a great impact on HCC translational medicine.展开更多
Summary： Recently, suppressor of cytokine signaling-3 （SOCS3） has been shown to be an inducible endogenous negative regulator of Janus kinase/signal transducers and activators of transcription （JAK/STAT） pathway ...Summary： Recently, suppressor of cytokine signaling-3 （SOCS3） has been shown to be an inducible endogenous negative regulator of Janus kinase/signal transducers and activators of transcription （JAK/STAT） pathway which is relevant in inflammatory response, while its functions in acute liver failure and HBV-induced acute-on-chronic liver failure （HBV-ACLF） have not been fully elucidated. In this study, we explored the role of SOCS3 in the development of mouse hepatitis virus strain 3 （MHV-3）-induced acute liver failure and its expression in liver and peripheral blood mononuclear cells （PBMCs） of patients with HBV-ACLF. Inflammation-related gene expression was detected by real-time PCR, immtmohistochemistry and Western blotting. The correlation between SOCS3 level and liver injury was studied. Our results showed that the SOCS3 expression was significantly elevated in both the liver tissue and PBMCs from patients with HBV-ACLF compared to mild chronic hepatitis B （CHB）. Moreover, a time course study showed that SOCS3 level was increased remarkably in the liver of BALB/cJ mice at 72 h post-infection. Pro-inflammatory cytokines, interleukin （IL）-1 β, IL-6, and tumor necrosis factor （TNF）-α, were also increased significantly at 72 h post-infection. There was a close correlation between hepatic SOCS3 level and IL-6, and the severity of liver injury defined by alanine aminotransferase （ALT） and aspartate aminotransferase （AST） levels, respectively. These data suggested that SOCS3 may play a pivotal role in the pathogenesis of MHV-3-induced acute liver failure and HBV-ACLF.展开更多
AIM:To evaluate the value of the hepatitis B virus(HBV) replication mouse model with regard to several aspects of the study of HBV biology.METHODS:To evaluate the HBV replication mouse model in detecting the efficacy ...AIM:To evaluate the value of the hepatitis B virus(HBV) replication mouse model with regard to several aspects of the study of HBV biology.METHODS:To evaluate the HBV replication mouse model in detecting the efficacy of anti-HBV agents,the interferon inducer polyinosinic-polytidylin acid(polyIC) and nucleotide analogues adefovir and entecavir were administered to mice injected with wild type pHBV4.1,and the inhibiting effect of these agents on HBV DNA replication was evaluated.To identify the model's value in a replication ability study of HBV drug-resistant mutants and a HBx-minus mutant,telbivudine resistance mutants(rtM204I,ayw subtype),adefovir resistance mutants(rtA181V + rtN236T,ayw subtype) and HBxminus mutants were injected respectively,and their corresponding HBV DNA replication intermediates in mouse liver were assessed.RESULTS:Compared with the wild type HBV replication mouse model without antiviral agent treatment,the HBV DNA replication intermediates of the polyICtreated group were decreased 1-fold;while in the entecavir-and adefovir-treated groups,the levels of HBV DNA replication intermediates were inhibited 13.6-fold and 1.4-fold,respectively.For the mouse models injected with telbivudine resistance mutant,adefovir resistance mutant and HBx-minus mutant,HBV DNA replication intermediates could still be detected,but the levels of HBV DNA replication intermediates of these mutants decreased 4.5-fold,5.6-fold and 2.9-fold respectively,compared with the mouse model with wild type HBV plasmid.CONCLUSION:The HBV replication mouse model we established was a useful and convenient tool to detect the efficacy of antiviral agents and to study the replication ability of HBV mutants in vivo.展开更多
AIM:To investigate the expression of the hepatitis B virus(HBV)1.3-fold genome plasmid(pHBV1.3)in an immortalized mouse hepatic cell line induced by SV40T-antigen(SV40T)expression.METHODS:Mouse hepatic cells were isol...AIM:To investigate the expression of the hepatitis B virus(HBV)1.3-fold genome plasmid(pHBV1.3)in an immortalized mouse hepatic cell line induced by SV40T-antigen(SV40T)expression.METHODS:Mouse hepatic cells were isolated from mouse liver tissue fragments from 3-5 d old Kunming mice by the direct collagenase digestion method and cultured in vitro.The pRSV-T plasmid was transfected into mouse hepatic cells to establish an SV40LT-immortalized mouse hepatic cell line.The SV40LT-immortalized mouse hepatic cells were identified and transfected with the pHBV1.3 plasmid.The levels of hepatitis B surface antigen(HBsAg)and hepatitis B e antigen(HBeAg)in the supernatant were determined by an electrochemiluminescence immunoassay at 24,48,72 and 96 h after transfection.The expressions of HBsAg and hepatitis B c antigen(HBcAg)in the cells were investigated by indirect immunofluorescence analysis.The presence of HBV DNA replication intermediates in the transfected cells and viral particles in the supernatant of the transfected cell cultures was monitored using the Southern hybridization assay and transmission electronic microscopy,respectively.RESULTS:The pRSV-T plasmid was used to immortalize mouse hepatocytes and an SV40LT-immortalized mouse hepatic cell line was successfully established.SV40LT-immortalized mouse hepatic cells have the same morphology and growth characteristics as primary mouse hepatic cells can be subcultured and produce albumin and cytokeratin-18 in vitro.Immortalized mouse hepatic cells did not show the characteristics of tumor cells,as alpha-fetoprotein levels were comparable(0.58±0.37 vs 0.61±0.31,P=0.37).SV40LTimmortalized mouse hepatic cells were then transfected with the pHBV1.3 plasmid,and it was found that the HBV genome replicated in SV40LT-immortalized mouse hepatic cells.The levels of HBsAg and HBeAg continuously increased in the supernatant after the transfection of pHBV1.3,and began to decrease 72 h after transfection.The expressions of HBsAg and HBcAg were observed in the pHBV1.3-transfected cells.HBV DNA replication intermediates were also observed at72 h after transfection,including relaxed circular DNA,double-stranded DNA and single-stranded DNA.Furthermore,a few 42 nm Dane particles,as well as many22 nm subviral particles with a spherical or filamentous shape,were detected in the supernatant.CONCLUSION:SV40T expression can immortalize mouse hepatic cells,and the pHBV1.3-transfected SV40T-immortalized mouse hepatic cell line can be a new in vitro cell model.展开更多
Transgenic mouse technology has enabled the investigation of the pathogenic effects, including those on development, immunological reactions and carcinogenesis, of viral genes directly in living organism in a real-tim...Transgenic mouse technology has enabled the investigation of the pathogenic effects, including those on development, immunological reactions and carcinogenesis, of viral genes directly in living organism in a real-time manner. Although viral hepatocarcinogenesis comprises multiple sequences of pathological events, that is, chronic necroinflammation and the subsequent regeneration of hepatocytes that induces the accumulation of genetic alterations and hepatocellular carcinoma(HCC), the direct action of viral proteins also play significant roles. The pathogenesis of hepatitis B virus X and hepatitis C virus(HCV) core genes has been extensively studied by virtue of their functions as a transactivator and a steatosis inducer, respectively. In particular, the mechanism of steatosis in HCV infection and its possible association with HCC has been well studied using HCV core gene transgenic mouse models. Although transgenic mouse models have remarkable advantages, they are intrinsically accompanied by some drawbacks when used to study human diseases. Therefore, the results obtained from transgenic mouse studies should be carefully interpreted in the context of whether or not they are well associated with human pathogenesis.展开更多
Mice infected with mouse hepatitis virus A59 (MHV-A59), an enveloped, positive-strand RNA Co-ronavirus, induce hepatitis, thymus involution, IgG2a-restricted hypergammaglobulinaemia, transaminase release and autoantib...Mice infected with mouse hepatitis virus A59 (MHV-A59), an enveloped, positive-strand RNA Co-ronavirus, induce hepatitis, thymus involution, IgG2a-restricted hypergammaglobulinaemia, transaminase release and autoantibodies (autoAb) to liver and kidney fumarylacetoacetate hy-drolase (FAH). Since Toll-like receptors (TLR) play a central role in innate immunity, we explored the effects of TLR3, 7 and 9 stimulation on MHV mouse infection. Thus, the animals were treated with Poly (I:C), Loxoribine and CpG, the respective TLR ligands. MHV-infected mice inoculated with Poly (I:C) had significant lower levels of plasma transaminases and Ig, anti-MHV Ab, and uric acid than MHV-infected animals, whereas autoAb to kidney tissue were observed. Loxoribine only produced a slight decrease of uric acid levels and serum Ig. CpG showed deleterious effects on MHV-infected mice, since survival of animals dramatically dropped to about 10%. AutoAb to murine tissues and uric acid release were not affected, whereas transaminases and anti-MHV Ab were slightly elevated. Besides, CpG administration produced a decrease of the high levels of serum Ig induced by the virus. Therefore, results indicated that TLR3 stimulation appeared to protect the animals against the viral infection, whereas CpG aggravated its signs. Loxoribine, the TLR7 ligand, did not show major effects.展开更多
Histone lysine methyltransferase EZH2 has been reported to be frequently overexpressed in hepatocellular carcinoma(HCC) tissues and associated with hepatocarcinogenesis.However,the exact mechanism of EZH2 up-regulatio...Histone lysine methyltransferase EZH2 has been reported to be frequently overexpressed in hepatocellular carcinoma(HCC) tissues and associated with hepatocarcinogenesis.However,the exact mechanism of EZH2 up-regulation in HCC has not been determined.In this study,we used murine hepatocyte AML12 cells to investigate the role of hepatitis B virus X protein(HBx) in regulating the expression of mEZH2.Western blot analysis demonstrated that the expression level of mEZH2 protein in AML12 cells was up-regulated by HBx in a dose-dependent manner.To further investigate the mechanism of mEZH2 overexpression,the 2500 bp regulatory sequence upstream from the first exon of the mEZH2 gene was amplified from AML12 genomic DNA and constructed into a luciferase reporter plasmid.The luciferase activity of the mEZH2 promoter significantly increased in AML12 cells co-transfected with HBx plasmid,and deleting the-486/-214 promoter region decreased HBx-induced mEZH2 promoter activation by nearly 50%.The-486/-214 region was then analyzed in the TRANSFAC 6.0 database and a typical E2F1-binding site was found.Mutation of this E2F1-binding site or knockdown of E2F1 expression by RNAi led to a dramatic decrease in HBx-induced activation of the mEZH2 promoter and mEZH2 overexpression in AML12 cells.These results provide evidence that HBx up-regulates mEZH2 expression by transactivating the mEZH2 promoter through E2F1 transcription factor,thereby providing new epigenetic evidence for the carcinogenic effect of HBx.展开更多
The narrow host range of infection supporting the long-term propagation of hepatitis B and C viruses is a major limitation that has prevented a more thorough understanding of persistent infection and the pathogenesis ...The narrow host range of infection supporting the long-term propagation of hepatitis B and C viruses is a major limitation that has prevented a more thorough understanding of persistent infection and the pathogenesis of chronic liver disease(CLD).With hepatitis B virus(HBV),this has been partially overcome by the discovery and characterization of HBV-like viruses in wild animals.With hepatitis C virus(HCV),related Flaviviruses have been used as surrogate systems for such studies.Independent work has developed various mouse strains for the transplantation of human hepatocytes,which are then susceptible to infection with HBV and HCV.Other laboratories have developed transgenic mice that express virus gene products and/or support virus replication.Some HBV transgenic mouse models develop fulminant hepatitis,acute hepatitis,or CLD following adoptive transfer,while others spontaneously develop hepatocellular carcinoma(HCC),as in human infections.Among HCV transgenic mice,most develop no disease,but acute hepatitis has been observed in one model,while HCC appears in another.Other mouse models include the introduction of xenographs that replicate HBV or HCV.Although mice are not susceptible to these viruses,their ability to support virus replication and to develop liver disease characteristic of human infections,provides new opportunities to study pathogenesis and develop novel therapeutics.展开更多
AIM: To develop a hepatocellular carcinoma (HCC) xenograft model for studying hepatitis C virus (HCV) replication in a mice, and antiviral treatment.METHODS: We developed a stable S3-green fluorescence protein (GFP) c...AIM: To develop a hepatocellular carcinoma (HCC) xenograft model for studying hepatitis C virus (HCV) replication in a mice, and antiviral treatment.METHODS: We developed a stable S3-green fluorescence protein (GFP) cell line that replicated the GFP-tagged HCV sub-genomic RNA derived from a highly efficient JFH1 virus. S3-GFP replicon cell line was injected subcutaneously into γ-irradiated SCID mice. We showed that the S3-GFP replicon cell line formed human HCC xenografts in SCID mice. Cells were isolated from subcutaneous tumors and then serially passaged multiple times in SCID mice by culturing in growth medium supplemented with G-418. The mouse-adapted S3-GFP replicon cells were implanted subcutaneously and also into the liver of SCID mice via intrasplenic infusion to study the replication of HCV in the HCC xenografts. The tumor model was validated for antiviral testing after intraperitoneal injection of interferon-α (IFN-α). RESULTS: A highly tumorigenic S3-GFP replicon cell line was developed that formed subcutaneous tumors within 2 wk and diffuse liver metastasis within 4 wk in SCID mice. Replication of HCV in the subcutaneous and liver tumors was confirmed by cell colony assay, detection of the viral RNA by ribonuclease protection assay and real-time quantitative reverse transcription polymerase chain reaction. High-level replication of HCV sub-genomic RNA in the tumor could be visualized by GFP expression using fluorescence microscopy. IFN-α cleared HCV RNA replication in the subcutaneous tumors within 2 wk and 4 wk in the liver tumor model. CONCLUSION: A non-infectious mouse model allows us to study replication of HCV in subcutaneous and metastatic liver tumors. Clearance of HCV by IFN-α supports use of this model to test other anti-HCV drugs.展开更多
基金Supported by Research grants from the Ministry of Science and Technology(MOST)in Taiwan,No.NSC99-2628-B-010-001-MY3,MOST 103-2321-B-010-003,MOST 103-2633-H-010-001,MOST 103-2633-B-400-002 and MOST104-3011-B-010-001a grant from the Ministry of Education,Aim for the Top University Plan
文摘The multifactorial and multistage pathogenesis of hepatocellular carcinoma(HCC)has fascinated a wide spectrum of scientists for decades.While a number of major risk factors have been identified,their mechanistic roles in hepatocarcinogenesis still need to be elucidated.Many tumor suppressor genes(TSGs)have been identified as being involved in HCC.These TSGs can be classified into two groups depending on the situation with respect to allelic mutation/loss in the tumors:the recessive TSGs with two required mutated alleles and the haploinsufficient TSGs with one required mutated allele.Hepatitis B virus(HBV)is one of the most important risk factors associated with HCC.Although mice cannot be infected with HBV due to the narrow host range of HBV and the lack of a proper receptor,one advantage of mouse models for HBV/HCC research is the numerous and powerfulgenetic tools that help investigate the phenotypic effects of viral proteins and allow the dissection of the dose-dependent action of TSGs.Here,we mainly focus on the application of mouse models in relation to HBV-associated HCC and on TSGs that act either in a recessive or in a haploinsufficient manner.Discoveries obtained using mouse models will have a great impact on HCC translational medicine.
基金supported by the grants from the National Science Foundation of China Advanced Program(No.NSFC81171558,NSFC81271808 and NSFC81030007)Innovation Team Development Plan of the Ministry of Education of China[No.IRT1131(2011)]National Twelfth-Five Years Project in Science and Technology of China(No.2013ZX10002-003)
文摘Summary： Recently, suppressor of cytokine signaling-3 （SOCS3） has been shown to be an inducible endogenous negative regulator of Janus kinase/signal transducers and activators of transcription （JAK/STAT） pathway which is relevant in inflammatory response, while its functions in acute liver failure and HBV-induced acute-on-chronic liver failure （HBV-ACLF） have not been fully elucidated. In this study, we explored the role of SOCS3 in the development of mouse hepatitis virus strain 3 （MHV-3）-induced acute liver failure and its expression in liver and peripheral blood mononuclear cells （PBMCs） of patients with HBV-ACLF. Inflammation-related gene expression was detected by real-time PCR, immtmohistochemistry and Western blotting. The correlation between SOCS3 level and liver injury was studied. Our results showed that the SOCS3 expression was significantly elevated in both the liver tissue and PBMCs from patients with HBV-ACLF compared to mild chronic hepatitis B （CHB）. Moreover, a time course study showed that SOCS3 level was increased remarkably in the liver of BALB/cJ mice at 72 h post-infection. Pro-inflammatory cytokines, interleukin （IL）-1 β, IL-6, and tumor necrosis factor （TNF）-α, were also increased significantly at 72 h post-infection. There was a close correlation between hepatic SOCS3 level and IL-6, and the severity of liver injury defined by alanine aminotransferase （ALT） and aspartate aminotransferase （AST） levels, respectively. These data suggested that SOCS3 may play a pivotal role in the pathogenesis of MHV-3-induced acute liver failure and HBV-ACLF.
基金Supported by The National Basic Research Program of China,No. 2007CB512902The National Basic Research Program of China, No. 2006CB504302Key Technologies Research of Sichuan Province,No. 2008SZ0039
文摘AIM:To evaluate the value of the hepatitis B virus(HBV) replication mouse model with regard to several aspects of the study of HBV biology.METHODS:To evaluate the HBV replication mouse model in detecting the efficacy of anti-HBV agents,the interferon inducer polyinosinic-polytidylin acid(polyIC) and nucleotide analogues adefovir and entecavir were administered to mice injected with wild type pHBV4.1,and the inhibiting effect of these agents on HBV DNA replication was evaluated.To identify the model's value in a replication ability study of HBV drug-resistant mutants and a HBx-minus mutant,telbivudine resistance mutants(rtM204I,ayw subtype),adefovir resistance mutants(rtA181V + rtN236T,ayw subtype) and HBxminus mutants were injected respectively,and their corresponding HBV DNA replication intermediates in mouse liver were assessed.RESULTS:Compared with the wild type HBV replication mouse model without antiviral agent treatment,the HBV DNA replication intermediates of the polyICtreated group were decreased 1-fold;while in the entecavir-and adefovir-treated groups,the levels of HBV DNA replication intermediates were inhibited 13.6-fold and 1.4-fold,respectively.For the mouse models injected with telbivudine resistance mutant,adefovir resistance mutant and HBx-minus mutant,HBV DNA replication intermediates could still be detected,but the levels of HBV DNA replication intermediates of these mutants decreased 4.5-fold,5.6-fold and 2.9-fold respectively,compared with the mouse model with wild type HBV plasmid.CONCLUSION:The HBV replication mouse model we established was a useful and convenient tool to detect the efficacy of antiviral agents and to study the replication ability of HBV mutants in vivo.
基金Supported by Jinan Science and Technology Bureau,Shandong Province,China,No.200705095-4
文摘AIM:To investigate the expression of the hepatitis B virus(HBV)1.3-fold genome plasmid(pHBV1.3)in an immortalized mouse hepatic cell line induced by SV40T-antigen(SV40T)expression.METHODS:Mouse hepatic cells were isolated from mouse liver tissue fragments from 3-5 d old Kunming mice by the direct collagenase digestion method and cultured in vitro.The pRSV-T plasmid was transfected into mouse hepatic cells to establish an SV40LT-immortalized mouse hepatic cell line.The SV40LT-immortalized mouse hepatic cells were identified and transfected with the pHBV1.3 plasmid.The levels of hepatitis B surface antigen(HBsAg)and hepatitis B e antigen(HBeAg)in the supernatant were determined by an electrochemiluminescence immunoassay at 24,48,72 and 96 h after transfection.The expressions of HBsAg and hepatitis B c antigen(HBcAg)in the cells were investigated by indirect immunofluorescence analysis.The presence of HBV DNA replication intermediates in the transfected cells and viral particles in the supernatant of the transfected cell cultures was monitored using the Southern hybridization assay and transmission electronic microscopy,respectively.RESULTS:The pRSV-T plasmid was used to immortalize mouse hepatocytes and an SV40LT-immortalized mouse hepatic cell line was successfully established.SV40LT-immortalized mouse hepatic cells have the same morphology and growth characteristics as primary mouse hepatic cells can be subcultured and produce albumin and cytokeratin-18 in vitro.Immortalized mouse hepatic cells did not show the characteristics of tumor cells,as alpha-fetoprotein levels were comparable(0.58±0.37 vs 0.61±0.31,P=0.37).SV40LTimmortalized mouse hepatic cells were then transfected with the pHBV1.3 plasmid,and it was found that the HBV genome replicated in SV40LT-immortalized mouse hepatic cells.The levels of HBsAg and HBeAg continuously increased in the supernatant after the transfection of pHBV1.3,and began to decrease 72 h after transfection.The expressions of HBsAg and HBcAg were observed in the pHBV1.3-transfected cells.HBV DNA replication intermediates were also observed at72 h after transfection,including relaxed circular DNA,double-stranded DNA and single-stranded DNA.Furthermore,a few 42 nm Dane particles,as well as many22 nm subviral particles with a spherical or filamentous shape,were detected in the supernatant.CONCLUSION:SV40T expression can immortalize mouse hepatic cells,and the pHBV1.3-transfected SV40T-immortalized mouse hepatic cell line can be a new in vitro cell model.
文摘Transgenic mouse technology has enabled the investigation of the pathogenic effects, including those on development, immunological reactions and carcinogenesis, of viral genes directly in living organism in a real-time manner. Although viral hepatocarcinogenesis comprises multiple sequences of pathological events, that is, chronic necroinflammation and the subsequent regeneration of hepatocytes that induces the accumulation of genetic alterations and hepatocellular carcinoma(HCC), the direct action of viral proteins also play significant roles. The pathogenesis of hepatitis B virus X and hepatitis C virus(HCV) core genes has been extensively studied by virtue of their functions as a transactivator and a steatosis inducer, respectively. In particular, the mechanism of steatosis in HCV infection and its possible association with HCC has been well studied using HCV core gene transgenic mouse models. Although transgenic mouse models have remarkable advantages, they are intrinsically accompanied by some drawbacks when used to study human diseases. Therefore, the results obtained from transgenic mouse studies should be carefully interpreted in the context of whether or not they are well associated with human pathogenesis.
文摘Mice infected with mouse hepatitis virus A59 (MHV-A59), an enveloped, positive-strand RNA Co-ronavirus, induce hepatitis, thymus involution, IgG2a-restricted hypergammaglobulinaemia, transaminase release and autoantibodies (autoAb) to liver and kidney fumarylacetoacetate hy-drolase (FAH). Since Toll-like receptors (TLR) play a central role in innate immunity, we explored the effects of TLR3, 7 and 9 stimulation on MHV mouse infection. Thus, the animals were treated with Poly (I:C), Loxoribine and CpG, the respective TLR ligands. MHV-infected mice inoculated with Poly (I:C) had significant lower levels of plasma transaminases and Ig, anti-MHV Ab, and uric acid than MHV-infected animals, whereas autoAb to kidney tissue were observed. Loxoribine only produced a slight decrease of uric acid levels and serum Ig. CpG showed deleterious effects on MHV-infected mice, since survival of animals dramatically dropped to about 10%. AutoAb to murine tissues and uric acid release were not affected, whereas transaminases and anti-MHV Ab were slightly elevated. Besides, CpG administration produced a decrease of the high levels of serum Ig induced by the virus. Therefore, results indicated that TLR3 stimulation appeared to protect the animals against the viral infection, whereas CpG aggravated its signs. Loxoribine, the TLR7 ligand, did not show major effects.
文摘Histone lysine methyltransferase EZH2 has been reported to be frequently overexpressed in hepatocellular carcinoma(HCC) tissues and associated with hepatocarcinogenesis.However,the exact mechanism of EZH2 up-regulation in HCC has not been determined.In this study,we used murine hepatocyte AML12 cells to investigate the role of hepatitis B virus X protein(HBx) in regulating the expression of mEZH2.Western blot analysis demonstrated that the expression level of mEZH2 protein in AML12 cells was up-regulated by HBx in a dose-dependent manner.To further investigate the mechanism of mEZH2 overexpression,the 2500 bp regulatory sequence upstream from the first exon of the mEZH2 gene was amplified from AML12 genomic DNA and constructed into a luciferase reporter plasmid.The luciferase activity of the mEZH2 promoter significantly increased in AML12 cells co-transfected with HBx plasmid,and deleting the-486/-214 promoter region decreased HBx-induced mEZH2 promoter activation by nearly 50%.The-486/-214 region was then analyzed in the TRANSFAC 6.0 database and a typical E2F1-binding site was found.Mutation of this E2F1-binding site or knockdown of E2F1 expression by RNAi led to a dramatic decrease in HBx-induced activation of the mEZH2 promoter and mEZH2 overexpression in AML12 cells.These results provide evidence that HBx up-regulates mEZH2 expression by transactivating the mEZH2 promoter through E2F1 transcription factor,thereby providing new epigenetic evidence for the carcinogenic effect of HBx.
基金sponsored by the National Institutes of Health(CA79512and AI-99-001)
文摘The narrow host range of infection supporting the long-term propagation of hepatitis B and C viruses is a major limitation that has prevented a more thorough understanding of persistent infection and the pathogenesis of chronic liver disease(CLD).With hepatitis B virus(HBV),this has been partially overcome by the discovery and characterization of HBV-like viruses in wild animals.With hepatitis C virus(HCV),related Flaviviruses have been used as surrogate systems for such studies.Independent work has developed various mouse strains for the transplantation of human hepatocytes,which are then susceptible to infection with HBV and HCV.Other laboratories have developed transgenic mice that express virus gene products and/or support virus replication.Some HBV transgenic mouse models develop fulminant hepatitis,acute hepatitis,or CLD following adoptive transfer,while others spontaneously develop hepatocellular carcinoma(HCC),as in human infections.Among HCV transgenic mice,most develop no disease,but acute hepatitis has been observed in one model,while HCC appears in another.Other mouse models include the introduction of xenographs that replicate HBV or HCV.Although mice are not susceptible to these viruses,their ability to support virus replication and to develop liver disease characteristic of human infections,provides new opportunities to study pathogenesis and develop novel therapeutics.
基金Supported by Funds received from the National Cancer Institute (CA127481,CA129776)Geyer Foundation,New York,Louisiana Cancer Research Consortium and Tulane Cancer Center
文摘AIM: To develop a hepatocellular carcinoma (HCC) xenograft model for studying hepatitis C virus (HCV) replication in a mice, and antiviral treatment.METHODS: We developed a stable S3-green fluorescence protein (GFP) cell line that replicated the GFP-tagged HCV sub-genomic RNA derived from a highly efficient JFH1 virus. S3-GFP replicon cell line was injected subcutaneously into γ-irradiated SCID mice. We showed that the S3-GFP replicon cell line formed human HCC xenografts in SCID mice. Cells were isolated from subcutaneous tumors and then serially passaged multiple times in SCID mice by culturing in growth medium supplemented with G-418. The mouse-adapted S3-GFP replicon cells were implanted subcutaneously and also into the liver of SCID mice via intrasplenic infusion to study the replication of HCV in the HCC xenografts. The tumor model was validated for antiviral testing after intraperitoneal injection of interferon-α (IFN-α). RESULTS: A highly tumorigenic S3-GFP replicon cell line was developed that formed subcutaneous tumors within 2 wk and diffuse liver metastasis within 4 wk in SCID mice. Replication of HCV in the subcutaneous and liver tumors was confirmed by cell colony assay, detection of the viral RNA by ribonuclease protection assay and real-time quantitative reverse transcription polymerase chain reaction. High-level replication of HCV sub-genomic RNA in the tumor could be visualized by GFP expression using fluorescence microscopy. IFN-α cleared HCV RNA replication in the subcutaneous tumors within 2 wk and 4 wk in the liver tumor model. CONCLUSION: A non-infectious mouse model allows us to study replication of HCV in subcutaneous and metastatic liver tumors. Clearance of HCV by IFN-α supports use of this model to test other anti-HCV drugs.
