Effects of wild- type and mutant hepatitis B virus X protein with deletion at C-terminus on cell proliferation in hepatocellular carcinoma

Objective To investigate the biological function of HBV X deletion mutation in N- terminus or C-terminus and its roles in carcinogenesis of hepatocellular carcinoma. Methods HBV X gene fragment encoding mutant HBV X protein with deletion 50 amino acid residues at N- terminus or C- terminus( HBxn or HBxc) was amplified from subtype amplification of HBV DNA by PCR. The purified HBxn and HBxc gene fragments were inserted into GFP expression vector,pEGFP- C1,to establish recombinant expression vector p GFP / HBxn and pGFP / HBxc. HepG2 cells were transfected with these recombinant constructs by lipofectamine reagent,and resistant clones expressing GFP- HBxn or GFP- HBxc fusion proteins were selected with G418 and observed under fluorescence microscope. The expression of deletion mutant HBV X gene was demonstrated by RT- PCR analysis. Growth rates and cell cycle were determined by MTT and flow cytometry analysis. The expression of p16 in these cells were analyzed by Western blot. Results RT- PCR analysis showed that HBxn and HBxc were expressed in GFP- HBxn and GFP- HBxc,respectively. MTT analysis showed that proliferation of HepG2 / GFP- HBx cells and HepG2 / GFP- HBxc cells were faster than that of HepG2,HepG2 / GFP and HepG2 / GFP- HBxn cells. Flow cytometry analysis showed that the percentage of G0/ G1 phase in HepG2 / GFP- HBx and HepG2 / GFP- HBxc cells were significantly decreased compared with HepG2 or HepG2 / GFP( P 0. 05). However,no significant differences were observed among Hep G2 /GFP- HBxn,HepG2 and HepG2 /GFP cells. Western blot analysis demonstrated that the levels of P16 in HepG2 / GFP- HBx and HepG2 / GFP- HBxc cells significantly decreased compared with HepG2 or HepG2 / GFP cells. However,level of p16 in HepG2 / GFP- HBxn cells were similar to that in HepG2 and HepG2 / GFP cells. Conclusion Wild- type and the C- terminus- deleted HBx promotes cell growth and cell cycle progression via down regulating p16 pathway. It is also indicated that the N- terminus of HBx is important in presenting biologic function of HBx in modulating cell cycle and carcinogenesis of hepatocellular carcinoma.