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Aim: To identify mutants of the hepatitis B virus (HBV) X (HBx) gene and investigate the effect of the natural mutant on liver cell proliferation. Methods: We identified natural mutants of the HBx gene from 188 sera and 48 tissues of Chinese patients infected with HBV by PCR, respectively. Based on the identification of the mutants of HBx gene, we cloned the fragments of the mutants into the pcDNA3 vector. The biological activities of the mutants were investigated. Results: We identified a natural mutant of the HBx gene with deletion from 382 to 401 base pairs from 3 sera out of 188 patients, which resulted in the expression deletion of the HBx protein from the 128th amino acid at the COOH terminal. The similar mutant with deletion from 382 base pair at the COOH terminal was identified from 5 cases of genomes out of 48 hepatocellular carcinoma tissues. Regarding the biological activities of the mutant, we found that the mutant of the HBx protein failed to induce apoptosis by transient transfection, but promoted proliferation of human liver immortalized L-O2 cells by stable transfection, compared with the wild-type HBx protein. The data showed that the proliferation of the mutant stably-transfected L-O2-X-Sera cells and fragment stably-transfected L-O2-X∆127 cells was enhanced by the BrdU incorporation assay and flow cytometry analysis. Luciferase reporter gene assay showed that the transcriptional activities of NF-κB, survivin, and human telomerase reverse transcriptase were upregulated, and Western blot analysis revealed that the expression levels of c-Myc and proliferating cell nuclear antigen (PCNA) were upregulated in the cells. Conclusion: Our findings suggest that the natural HBx mutant truncated 27 amino acids at the COOH terminal promotes cell proliferation.

Aim:To demonstrate the gene expression profiles mediated by hepatitis B virus X protein (HBx), we characterized the molecular features of pathogenesis associated with HBx in a human liver cell model. Methods: We examined gene expression profiles in L-O2-X cells, an engineered L-O2 cell line that constitutively expresses HBx, relative to L-O2 cells using an Agilent 22 K human 70-mer oligonucleotide microarray representing more than 21,329 unique, well-characterized Homo sapiens genes. Western blot analysis and RNA interference (RNAi) targeting HBx mRNA validated the overexpression of proliferating cell nuclear antigen (PCNA) and Bcl-2 in L-O2-X cells. Meanwhile, the BrdU incorporation assay was used to test cell proliferation mediated by upregulated cyclooxygenase-2 (COX-2). Results:The microarray showed that the expression levels of 152 genes were remarkably altered; 82 of the genes were upregulated and 70 genes were downregulated in L-O2-X cells. The altered genes were associated with signal transduction pathways, cell cycle, metastasis, transcriptional regulation, immune response, metabolism, and other processes. PCNA and Bcl-2 were upregulated in L-O2-X cells. Furthermore, we found that COX-2 upregulation in L-O2-X cells enhanced proliferation using the BrdU incorporation assay, whereas indomethacin (an inhibitor of COX-2) abolished the promotion. Conclusion: Our findings provide new evidence that HBx is able to regulate many genes that may be involved in the carcinogenesis. These regulated genes mediated by HBx may serve as molecular targets for the prevention and treatment of hepatocellular carcinoma.