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

YAO Xue-bing; YANG Lin; ZHU Jian-yun; MAI Li; CHONG Yu-tian

Abstract：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 expres-sion vector, pEGFP-C1, to establish recombinant expression vector pGFP/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 G 418 and observed under fluorescence microscope .The expression of dele-tion 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 G 0/G1 phase in HepG2/GFP-HBx and HepG2/GFP -HBxc cells were significantly decreased compared with HepG 2 or HepG2/GFP (P<0.05).However, no significant differences were observed among HepG 2/GFP-HBxn,HepG2 and HepG2/GFP cells.Western blot analysis demonstrated that the levels of P 16 in HepG2/GFP-HBx and HepG2/GFP-HBxc cells sig-nificantly 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 p 16 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 .