Effects Of HCV NS2 And NS3/4A Proteins On The Genomic Stability Of Human Hepatoma Huh7 Cells

Objective:Hepatocellular carcinoma(HCC)is one of the most common malignancies leading to high morbidity and mortality in China.Chronic infection of hepatitis C virus(HCV)is a major risk factor in the development of the HCC,however the molecular mechanisms are not well understood.In this study,eukaryotic expression plasmids of pCMV-tag2B-NS2 and pLV-Green-NS3/4A were transfected into Huh7 cells to investigated the effects of HCV NS2 and NS3/4A proteins on cellular genomic stability.It will provide a novel way about the pathogenesis of Hepatitis C virus-induced hepatocellular carcinoma.Methods:1.Construction of eukaryotic expression plasmid:HCV NS2 gene was amplified by polymerase chain reaction(PCR)from pJFH1(cDNA of HCV 2a genotype)and eukaryotic expression plasmid of pCMV-tag2B-NS2 was constructed by double enzyme digestion,then identified by enzyme digestion and DNA sequencing.2.Westernblot:TherecombinantplasmidsofpCMV-tag2B-NS2and pLV-Green-NS3/4A were transfected into Huh7 cells.The influence of HCV NS2 and NS3/4A proteins on cell apoptosis and cellular response to DNA damage were detected by DAPI stains and Western blot,respectively.3.Immunofluorescence:After transfected pLV-Puro and pLV-Puro-NS3/4A into U2OS cells,the expression level ofγ-Tubulin andα-Tubulin were identified by immunofluorescence.4.Flowcytometryanalysis:TheplasmidsofpCMV-tag2B-NS2and pLV-Green-NS3/4A were transfected into Huh7 cells.The influence of HCV NS2 and NS3/4A proteins on cell cycle progression was analyzed by flow cytometry analysis.5.MTT assays:After transfected pLV-Green and pLV-Green-NS3/4A into Huh7 cells,then treated with Taxol or Nutlin-3.The changes in proliferation rate of NS3/4A-expressing cells were reflected by MTT colorimetric assays.6.Data analysis:Data statistics was analyzed by SPSS19.0 software,measurement data was represented by mean±standard deviation,the rate comparison between groups used chi-square test,P<0.05 was considered statistically significant.Results:1.The recombinant plasmid of pCMV-tag2B-NS2 was constructed successfully.2.The apoptosis rates of NS2-expressing cells and control cells were(15.33±0.88)%and(4.66±0.88)%,respectively(P<0.05).The cell cycle progression of NS2-expressing cells was similar with control cells(?~2=0.17,P>0.05).The expression levels ofγH2AX、CHK1 and RPA32 in NS2-expressing cells were significantly higher than those of the control cells.3.Compared with the control cells,phosphorylation of NBS1,CHK1 and RPA32 were decreased after Camptothecin(CPT)treatment in NS3/4A-expressing cells.4.TherewerecentrosomeamplificationandabnormaldivisioninHCV NS3/4A-expressing Huh7 cells(P<0.05).The proliferation activity decreased significantly treated with Taxol in NS3/4A-expressing Huh7 cells(P<0.05).5.The expression of NS3/4A protein arrested cell cycle progression in G2/M phase(?~2=6.21,P<0.05).Conclusion:1.HCV NS2 protein promotes Huh7 cells apoptosis,but does not affect the cell cycle progression.2.HCV NS2 protein induces DNA damage checkpoint branch by activating CHK1phosphorylation.3.HCV NS3/4A protein induces DNA damage and interference of DNA damage response mediated by ATM and ATR kinases.4.HCV NS3/4A protein induces centrosome amplification and aberrant mitosis,improving sensitivity to Taxol in Huh7 cells.5.HCV NS3/4A protein can induce DNA damage but inhibit its repair,resulting in genomic instability.