Effects Of Hepatitis C Virus Genotype1b Core Protein And NS4B Protein On HepG2Cell Apoptosis

Objective:The purpose of this study was to investigate the effects of hepatitis C virus (HCV)core protein (Core) and nonstructural protein4B(NS4B) on HepG2cell apoptosis, andto explore the relationship of1b genotype HCV Core,NS4B and cell apoptosis,itspossible mechanism.Methods:The recombination vectors of HCV1b type Core(pcDNA3.1(-)-Core) andNS4B(pcDNA3.1(-)-NS4B) were transfected into HepG2cells by Lipofectaminerespectively,meanwhile the HepG2cells transfected with pcDNA3.1(-) and nontreatedHepG2cells were used as control(the four cell groups were named GrouppcDNA3.1(-)-Core,Group pcDNA3.1(-)-NS4B,Group pcDNA3.1(-) and Group blankcontrol respectively).After48h transfection,the expression of mRNA and protein ofHCV Core and NS4B were examined by RT-PCR and Western blotting analysisrespectively. With or without TNF-α treatment, growth of each cell group wereexamined by MTT analysis;Flow cytometry analysis was used to evaluate apoptosis ofeach cell group;and to evaluated the relative expression mRNA and protein of CleavedCaspase-3and XIAP in each cell group, RT-PCR and Western blotting analysis wereused.Results:1.The corresponding proteins were successfully expressed in the HepG2cellswhich were transiently transfected with the recombination vectors pcDNA3.1(-)-Coreand pcDNA3.1(-)-NS4B through RT-PCR and Western blotting analysis respectively.2.Using RT-PCR and Western blotting analysis,we found that the relativeexpression of mRNA and protein of Cleaved Caspase-3were lower in GroupspcDNA3.1(-)-Core, pcDNA3.1(-)-NS4B than those in the Group pcDNA3.1(-) and Group blank control(p<0.01),and the relative expression of mRNA and protein ofXIAP were higher in Groups pcDNA3.1(-)-Core, pcDNA3.1(-)-NS4B than those in theGroup pcDNA3.1(-) and Group blank control(p<0.01).The relative expression ofmRNA and protein of Cleaved Caspase-3in Group pcDNA3.1(-)-Core,GrouppcDNA3.1(-)-NS4B which were treated with TNF-α were higher than those in theGroup pcDNA3.1(-) and Group blank control(p<0.01),while the relative expression ofmRNA and protein of XIAP were lower than those in the Group pcDNA3.1(-) andGroup blank control(p<0.01).Compared with the four groups which were untreatedwith TNF-α,the relative expression of mRNA and protein of Caspase-3in the fourgroups treated with TNF-α increased significantly(p<0.01),the relative expression ofmRNA and protein of XIAP were lower in Groups pcDNA3.1(-)-Core,pcDNA3.1(-)-NS4B (p<0.01),and the relative expression of mRNA and protein ofXIAP were higher in the Group pcDNA3.1(-) and Group blank control (p<0.01).Therewere no statistical significance among the other groups (P>0.05).3.At one to five days after the transfection, MTT analysis evaluated that the rateof cells death were less in Groups pcDNA3.1(-)-Core, pcDNA3.1(-)-NS4B than thosein the Group pcDNA3.1(-) and Group blank control(p<0.01). The rate of cells death inGroup pcDNA3.1(-)-Core,Group pcDNA3.1(-)-NS4B treated with TNF-α were higherthan those in the Group pcDNA3.1(-) and Group blank control(p<0.01).Compared withthe four groups untreated with TNF-α,the rate of cells death in the four groups treatedwith TNF-α increased significantly(p<0.01).There were no statistical significanceamong the other groups(P>0.05).4.Flow cytometry indicated that the rate of cells apoptosis were lower in GroupspcDNA3.1(-)-Core, pcDNA3.1(-)-NS4B than those in the Group pcDNA3.1(-) andGroup blank control(p<0.01).The rate of cells apoptosis in GrouppcDNA3.1(-)-Core,Group pcDNA3.1(-)-NS4B treated with TNF-α were higher thanthose in the Group pcDNA3.1(-) and Group blank control(p<0.01).Compared with thefour groups untreated with TNF-α,the rate of cells apoptosis in the four groups treatedwith TNF-α increased significantly(p<0.01).There were no statistical significanceamong the other groups (P>0.05). Conclusions:1.HCV Core and NS4B can down-regulate the expression of Cleaved Caspase-3and up-regulate the expression of XIAP in HepG2cells.2.Under induction of the TNF-α,HCV Core and NS4B can up-regulate theexpression of Cleaved Caspase-3and down-regulate the expression of XIAP in HepG2cells,thus enhance TNF-α-induced cell death.