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Aim: To investigate the effects of p16 gene on biological behaviours in hepatocellular carcinoma cells., Methods: HCC cell lines SNU-449 and HepG2.2.15 were infected respectively by a replication defective, recombinant retrovirus capable of producing a high level of p16 protein expression (pCLXSN-p16). G418 resistant stable P16 protein expression cell lines were selected. And the biological behaviours of the p16 gene transfected HCC cells were observed., Results: Initial in vitro experiments in HCC cell line SNU-449 with loss of p16 protein expression demonstrated the pCLXSN-p16 treatment significantly inhibited cell growth. But there was no treatment effect when the pCLXSN-p16 was used in another HCC cell line HepG2.2.15 which has positive p16 protein expression. Subsequent study in a nude mouse model demonstrated that the p16 gene transfected SNU-449 had a lower succeeding rate in the first time establishment of tumors and grew more slowly in the nude mice when compared with non-transfected SNU-449. Moreover, the nude mice inoculated with transfected SNU-449 had a longer surviving time than those inoculated with non-transfected SNU-449., Conclusion: Our results show that the p16INK4a gene transfer can inhibit the proliferation and reduce the invasion ability of hepatocellular carcinoma.