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Objective:To investigate the role of protein phosphatase 4 catalytic subunit (PP4C) in regulating hepatitis B virus X protein (HBx) levels and its effects on the biological functions of HBx, thus to provide a potential therapeutic targets for hepatitis B virus (HBV)-related hepatocellular carcinoma.Methods:In vivo and in vitro interactions between HBx and PP4C were analyzed by co-immunoprecipitation (Co-IP) and GST pull-down assay. Recombinant plasmids of PP4C and HBx were co-transfected with Lipofectamine 3000 reagents into hepatoma cells to detect the protein levels of HBx by Western blot. The half-life of HBx in the transfected cells treated with cycloheximide (CHX) were detected. The phosphorylation assay was used to evaluate the effects of PP4C on HBx phosphorylation. CCK8 assay, wound healing assay and Matrigel invasion chamber assay were used to analyze the effects of PP4C on the biological functions of HBx. Results:PP4C interacted with HBx in vivo and in vitro. PP4C overexpression significantly increased the protein level and stability of HBx and the phosphorylation assay confirmed that PP4C overexpression decreased the serine phosphorylation of HBx in hepatoma cells. PP4C overexpression enhanced the migration and invasion of hepatoma cells, but had no significant effects on the proliferation. Conclusions:The interactions between HBx and PP4C promoted the stability of HBx and ultimately enhanced the migration and invasion of hepatoma cells, and the mechanisms might be related to the decrease of HBx serine phosphorylation by PP4C. This study provided a theoretical basis for further investigation of the pathogenic mechanisms of HBx, and targeting PP4C and HBx interaction might provide insights for developing novel treatment for HBV-related hepatocellular carcinoma.
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Objective:To investigate the effects of the interaction between ubiquitin-specific peptidase 22 (USP22) and hepatitis B virus X protein (HBx) on the protein level and the biological function of HBx.Methods:The interactions between HBx and USP22 were analyzed by GST pull-down, co-immunoprecipitation assay and confocal laser scanning assay. USP22 recombinant plasmids or specific siRNA were transiently co-transfected with HBx plasmids. Western blot were used to detect the protein level of HBx. The half-life and degradation pathway of HBx in the transfected cells treated with cycloheximide (CHX) or proteasome inhibitor MG132 were detected. In vivo ubiquitination assay was used to detect the ubiquitination of HBx with USP22 overexpression. Moreover, dual-luciferase reporter assay and colony formation assay were used to analyze the effects of USP22 on the biological function of HBx. Results:USP22 could interact with HBx in vivo and in vitro. USP22 significantly increased the stability of HBx and inhibited the proteasome-mediated degradation of HBx protein by reducing the ubiquitination of HBx, thereby enhancing the biological function of HBx. Conclusions:USP22 inhibited HBx protein degradation through ubiquitin-dependent proteasome pathway, thus enhancing the stability and biological function of HBx.
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To investigate the TGFβ1-induced epithelial-mesenchymal transition (EMT) of Huh7 hepatoma cells caused by interaction of hepatitis B spliced protein (HBSP) with transforming growth factor beta-1-induced transcript 1 protein (TGFβ31I1),coding region of HBSP was cloned into lentiviral expression vector.Huh7 hepatoma cells were infected by recombinant lentivirus packaged in 293T cells.Stable cell lines expressing HBSP or control cells were selected by puromycin.Cells were incubated with 5 ng/mL TGFβ1 for 24 h,and observed under contrast-phase microspcope.Then the whole cell lysates were collected for western blot analysis using specific antibodies against EMT markers including E-cadherin,N-cadherin,Claudin-1 and β-catenin.To evaluate the effects of HBSP-TGFβ1I1 interaction on EMT,TGFβ1-induced EMT marker transition,as well as cell invasion and migration were explored after knocking down of TGFβ1I1 by siRNA.Results showed that Huh7 cell lines expressing HBSP (Huh7-HBSP flag-HIV) and control cell lines (Huh7-flag-HIV) were successfully established.Huh7-HBSP flag-HIV cells lost their pebble-like shape and tight cell-cell adhesion and transformed into the mesenchymal-like cells in the presence of TGFβ1.Decreased expression level of epithelial marker of E-cadherin,Claudin-1,β-catenin,increased expression level of mesenchymal marker of N-cadherin,and enhanced migration and invasion abilities were observed in Huh7-HBSP-flag-HIV cells as compared to the control cells.Moreover,the changes of EMT markers and metastasis abilities of Huh7-HBSP-flag-HIV cells could be reversed when TGFβ111 was knocked down by siRNA.In conclusion,HBSP could promote hepatoma cell migration and invasion by triggering EMT via interaction with TGFβ111.Our findings highlight new insights for HBSP-induced HCC progression.
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Objective To screen the liver cell proteins interacting with the novel protein encoded by the 2.2 kb singly spliced variant of hepatitis B virus genome. Methods The splicing-specific gene TPss generated by the 2.2 kb singly spliced variant of HBV genome was amplified by PCR and cloned into the bait vector pGBKT7. After exclusion of self-activatian capacities of TPss protein, a two-hybrid library screening was performed using a pre-transformed human liver cDNA library to screen the liver cell proteins interacting with TPss. Mammalian two-hybrid assay was also done to further confirm the interactions between the bait and prey proteins in Huh7 and HepG2 hepatacytes. Results TPss gene with the size of 336 bp was successfully amplified and cloned, and the TPss protein expressed well in AHI09 yeast cells. Four liver cell proteins interacting with TPss, i. e. , cathepsin B, epoxide hydrolase 1, cathepsin D and fibrinogen gamma chain, were screened by yeast two-hybrid assay and further confirmed by mammalian two-hybrid assay. Conclusion TPss could interact with liver cell proteins.
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Objective To elucidate the genome organization of small deletion mutants of hepatitis B virus(HBV).Methods Amplified the HBV genomes by polymerase chain reaction from the serum of the patients with chronic hepatitis B and cloned the small HBV DNA less than 1 kb,then sequenced and analyzed the gene organization of these small deletion mutants of HBV.Results Totally one hundred and twenty-four small deletion mutants of HBV genomes categorized to sixty-four types were obtained and classified into three groups according to the criteria of the characteristics of gene organization,for example,spliced variants,regular deletion mutants and the deletion mutants with an internal poly (dA).All of these isolated mutants shared some common features as the deletion in coding regions and regulatory elements,66% of the mutants retained the cis elements crucial for the viral replication and encapsidation,while 48% retained the X region.Conclusions Small deletion mutants of HBV are commonly detected in the serum from chronic hepatitis B patients,the characteristic structure of such mutants implies that they might be closely co-related with the pathogenicity of HBV.The exact mechanisms need further study yet.
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Objective To investigate the effects of the novel protein,TPds encoded by the 2.2 kb doubly spliced variant of hepatitis B virus(HBV)genome on the regulatory elements of HBV.Methods HBV promoters and enhancers were amplified by PCR and respectively cloned into the plasmid pGL3-basic to construct the recombinant firefly luciferase reporter vectors including pGL3-BCP(harboring basal core promoter),pGL3-CP1601(core promoter with enhancer Ⅱ),pGL3-XP(minimal X gene promoter),pGL3 XP1071(X gene promoter with enhancer Ⅰ),pGL3-SP1(promoter of large surface antigen)and pGL3-SP2 (promoter of middle surface antigen).The recombinant reporter plasmids were respectively co-transfected with either the pcDNA3.1/HisC-TPds(TPd8 expression plasmid)or pcDNA3.1/HisC(empty control)into Huh7 hepatocytes by FuGENE6 transfection reagent.Cells were lysed 48 h post transfection,the intracellulax luciferase activities were monitored and calculated by SPSS11.5 software.Results As compared with the empty control of pcDNA3.1/HisC.the intracellular luciferase activities were elevated when the Huh7 hepatocytes were co-transfected by pcDNA3.1/HisC-TPds with one of the recombinant reporter plasmids as pGL3-CP1601,pGL3-XP1071,pGL3-SP1,or pGL3-SP2,while no changes with pGL3-BCP or pGL3-XP.Conduslon TPds could transactivate the HBV regulatory elements a8 promoter SP1,SP2.and enhancer Ⅰ,Ⅱ,while no influences on basal core promoter and minimal X gene promoter.
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Objective To investigate the anti-IFN-α effects of the novel protein TSR'r' encodedby the 458 nt-1308 nt spliced variant of hepatitis B virus genome,and to determine its functional domaias.Methods the TSR'r' gene(originated from open reading frame of HBV DNA polymerase,T represents terminal protein region,S represents the Spacer region,R'represents the truncated reverse transcriptase region,and r'represents the truncated RNaseH region)of the 458 nt-1308 nt spliced variant of HBV genome and its deletants were amplified by PCR and were cloned into the pcDNA3.1/HisC vector.The recombinant vector was transfected into Huh7 hepatocytes individually by FuGENE6 transfection reagent,and the expression of the fusion protein was detected by Western blot.Huh7 hepatocytes were co-transfected with p6 16CAT and the recombinant vector harboring either TSR'r'or the related deletant,and treated with IFN-α 2a 48 h post transfection.After 24 h stimulating.the cells were lysed and the intracellular CAT value was calculated.All data were processed with One-way analysis of variance(ANOVA).Resuits Recombinant vectors harboring either the TSR'r'gene or related deletant were constructed successfully,and the fusion proteins were expressed well in Huh7 cells.When Huh7 hepatocytes were co-transfected with p6-16CAT and TSR'r' recombinant.the intracellular CAT values reduced gradually as paralleled with the increasing amount of TSR'r'recombinant.Furthermore,as compared with the empty vector,intracellular CAT values also decreased significantly when the Huh7 cells co-transfected with recombinant harboring TP plus Spacer regions,while any of the other deletants(harboring either TP or Spacer region or neither)showed no significant difference.Conclusion The novel protein encoded by the 458 nt-1308 nt spliced variant of hepatitis B virns genome suppressed the response of Huh7 hepatocytes to IFN-α.and the N-terminal TP plus Spacer region was the functional domain of the protein for anti-IFN-α effects.