Effect Of Interaction Of Hepatitis B Virus X Protein And HDaxx On Apoptosis Of HepG2 Cells

Objective: To provide the experimental basis for further studying the effect on oncogenic mechanism of hepatitis B virus (HBV )X protein (HBx), this study was designed to identify the interaction of HBx with hDaxx by yeast two-hybrid system and to explore its effect on apoptosis of HepG2 cells.Methods:(1) Construction of pGADT7-HBx: Polymerase chain reaction(PCR) was used to amplify HBV X gene which was built in EcoRI and XhoI restriction enzyme sites. The fragment was directly ligated into pGADT7 vector. Then the recombinant was transformed into E.coli DH5αand identified by restriction enzyme analysis and sequencing.(2) Construction of pGBKT7-hDaxx: After the restriction enzyme reaction of pGBDU-C1/hDaxx and pGBKT7,the fragment of hDaxx gene was subcloned into bait vector pGBKT7. Then the recombinant was transformed into E.coli DH5αand identified by restriction enzyme analysis.(3) Yeast two-hybrid assay: Four groups of different plasmids were divided for transforming into yeast (AH109) respectively. Group A was pGADT7 and pGBKT7; group B was pGADT7-Hbx and pGBKT7-hDaxx; group C was pGADT7-T and pGBKT7-Lam; group D was pGADT7-T and pGBKT7-p53. The transformants were plated on SD/-Trp-Leu plates and incubated at 30°C for 2 to 4 days. The positive clones were streaked to the SD/-Trp-Leu-His plates or SD/-Trp-Leu-His-Ade plates, and then incubated at 30°C for 2 to 4 days. The expression of hDaxx and X protein were detected by Western blot in positive colonies of group B .(4) Flow cytometry assay: The expression plasmids of pcDAN3.1-HBx was stably transfected into HepG2 cells, then 15μg,30μg and 45μg pcDAN3.1-hDaxx were respectively transfected into these cells. The groups of HepG2X cells and that transfected by pcDNA3.1 were arranged as control. 5-FU was used to induce apoptosis for 36h. Every group cells were collected, respectively, and fixed in 75% ethanol at 4℃overnight. The cells were stained by propidium iodide (PI).Then apoptosis of every group was detected by FCM assay. Apoptotic rate of every group was analyzed by SPSS13.0.Results:(1) The size and sequence of HBV X fragment in pGADT7-HBx was identical with that in GeneBank(Pubmed NC_U95551)using restriction enzyme analysis and sequencing.(2) The gene of hDaxx was subcloned into the expression vector pGBKT7 by restriction enzyme analysis.(3) There were white colonies on SD/-Trp-Leu plates of the four groups. But only the yeast of group B and D could grow on SD/-Trp-Leu-His plates and SD/-Trp-Leu-His-Ade plates. The expressions of hDaxx and HBx protein were detected in positive colonies of group B by western blot.(4) The expression plasmids of pcDAN3.1-HBx was stably transfected into HepG2 cells. The expression of HBx were detected by Western blot. The apoptosis rate of HepG2 cells treated with 5-FU was much lower than that of HepG2 cells without 5-FU (P<0.01); The apoptosis rate of cells expressing HBx stably was much lower than that of HepG2 cells(P<0.01); And the apoptosis rate of the groups of co-transfected pcDAN3.1-hDaxx were degraded but unrelated with the dose of pcDNA3.1-hDaxx.Conclusion:(1) The recombinant plasmid of pGADT7-HBx and pGBKT7-hDaxx were successfully constructed, and the two protein could express correctly in yeast AH109.(2) HBx could interact with hDaxx in yeast two-hybrid system.(3) A gene transfered HCC cell line HepG2X, which could stably express HBx , was established successfully.(4) HBx inhibited the apoptosis of HepG2X cells induced by 5-FU ; The over-expression of hDaxx could made the apoptosis rate lower. It seemed that HBx might inhibit the apoptosis of HepG2 cells by interacting with hDaxx.