[Function of one novel gene identified by SSH PCR differentially expressed in HBX transfected HepG2 cells]

To clone full length differentially expressed genes which are related with HBxAg.

HepG2-cells were infected with prepared recombinant retroviruses encoding the X antigen. The differences in gene expression between HepG2 x and HepG2Cat cells were evaluated by suppression subtractive hybridization and PCR. In situ hybridization (ISH) and Northern blot analysis were carried out to screen the differentially expressed genes. The full length cDNA clone of the gene was obtained by 5' and 3' rapid amplification of cDNA ends(race) PCR. HepG2 cells transiently transfected with the new full length gene were subjected to fluorescence activated cell sorting (FACS) analysis for DNA content. HepG2 cells stably transfected with the new full length gene were tested for anchorage independent growth in soft agar and for tumorigenicity in nude mice.

The expression of multiple genes were turned on (8) or off (2) in HepG2X compared to HepG2CAT cells. One differentially expressed gene C2, the human homology of Sui1, encoded a translation initiation factor whose expression was suppressed by X antigen in HepG(2) cells. The full length of this gene was 1.35 kb, which encoded a small protein of 113 amino acids. Introduction of C2 into HepG2 cells could inhibit cell growth in culture, in soft agar, and partially inhibit tumor formation in nude mice. Cells transfected with pcDNA3-HBx showed little or no detectable C2, which was consistent with the suppression of this protein in the presence of HBxAg. C2 was also expressed in nontumor liver, but not in tumor cells from patients with hepatocellular carcinoma.

HBX can regulate the expression of genes whose products may be positive or negative regulators of cell growth. Our work for the first time demonstrates that the mechanism of DNA virus associated carcinogenesis involves altered patterns of gene expression regulated at the level of translation initiation.