HBV infection is a major factor contributing to the development of HCC in China and the mutation in HBx plays an important role in this process[2,19–22
]. However, analysis of HBx sequence in tumor tissues of HCC patients from mainland China has not been done[14,16,22,23
]. A previous study of sera from 67 HCC patients from Taiwan indicates that 52% of samples contain HBx mutations[23
]. Our present study showed that the frequency of the HBx gene mutation in either tumor tissues or the corresponding non-cancerous liver tissue samples of HCC is very high, 57.9% and 94.7%, respectively. The most frequent spots of mutation identified are 67aa (8/20) and 127aa (6/20). The hot spots reported by other groups are nt. 382-389 (codons 128-130) in HCC samples collected from Qidong, China[14
], nt. 204 and 260-264 (codons 68 and 87-88) in HCC samples from Hong Kong[16
] and nt. 93 (codon 31) in HCC samples from Taiwan[23
]. These suggested that the HBx may have its own distinguished patterns of mutation in different geographic regions. As was reported by Chen GG et al, the biological consequence may be the same in all these regions[16
The most important finding of this study was the identification of HBx deletion mutation in our samples. HBx deletion was detected in 16 HCC samples, and 14 exhibited the COOH-terminal deletion of HBx in HCC tissues. A COOH-terminal deletion in the HBx gene was found in 5 of 9[24
] and 5/6 HCCs[15
]. There may be three regions of the X gene essential for the transactivation function of the X protein (at codons 46-52, 61-69 and 132-139)[14,25
]. All of these results suggest that HBx mutants with a COOH-terminal deletion were significantly correlated with the development and progression of HCC.
Although the mechanism involved in pathogenesis of HBx mutants with a COOH-terminal deletion remains largely unknown, some studies showed that HBx deleted mutants isolated from tumor tissues abrogated both the transactivation and antiproliferative effects of wild type HBx[24
]. When HBx deleted mutant plasmids were transfected to murine and human cell lines, a strongly increased colony formation, accelerated cell cycle progression, and synergetically promoted ras and myc transforming capacity were confirmed[7,24,26
]. Therefore, a COOH-terminal deletion may alter the balance of HBx functional domains in regulating cell proliferation and apoptosis, viability, and transformation. In addition, as previously reported[15,27
], we also noted the coexistence within the same tumor cells of full-length and COOH-terminally deleted HBx sequences, encoded by free or integrated HBV genome sequences. Although we do not exclude the coexistence within the same tumor cells of full-length and COOH-terminally deleted HBx sequences encoded by free or integrated HBV genome, we are investigating the biological implication of the COOH-terminally truncated HBx sequences.
Integration of HBV DNA was found in HCC at GA-rich region of 17p13 locus in our study. We have eliminated the mispriming of HBV primer, and identified a specific integration of HBV DNA . This region includes the p53 gene, which is bound to and inactivated by HBxAg prior to tumor formation and then lost during tumor progression[19
]. This region also encodes microRNAs-22, -132, -195, -212[19
]; ubiquitin-conjugating enzyme E2G 1 and ubiquitin specific protease[28
]. We have also noted that most of the samples contained COOH-terminally deleted mutants of the HBx, and the specific integration of HBx was more frequent in HCC than in the corresponding non-cancerous liver tissues, and HBV integration including HBx, occurred in partial HCC tissues by Southern blot. These changes, to the best of our knowledge, have not been described elsewhere. Almost all of the HBV-associated HCCs harbor chromosomally integrated HBV DNA sequences, including chromosome 17p12-13[4,19,29,30
]. HBV integration can induce deletions in the host chromosome at the integration site[19
]. It was also recently confirmed that HBV insertion into cellular genes is a frequent event and that integration can occur in genes regulating cellular signal transduction cascades, proliferation control and cell viability[7,19
]. Thus, the putative HBV-specific integration sites, the biological impacts and the process with functional genomics of HBV associated HCCs need further studies.
In conclusion, HBx deletion, especially the COOH-terminal deletion of HBx, is a frequent event in HBV-associated HCC tissues. HBV integration including HBx, occurred in partial HCC tissues. This supports the hypothesis that the deletion and probably integrated forms of the HBx gene may be implicated in liver carcinogenesis.