COOH-terminal deletion of HBx gene is a frequent event in HBV-associated hepatocellular carcinoma

Abstract

Aim: To investigate the hepatitis B virus (HBV)x gene (HBx) state in the tissues of HBV-related hepatocellular carcinoma (HCC) in Chinese patients and whether there were particular HBx mutations.

Methods: HBx gene was amplified and direct sequencing was used in genomic DNA samples from 20 HCC and corresponding non-cancerous liver tissues from HBsAg-positive patients. HBV DNA integration and HBx deleted mutation were validated in 45 HCC patients at different stages by Southern blot analysis and polymerase chain reaction methods.

Results: The frequencies of HBx point mutations were significantly lower in HCC than their corresponding non-cancerous liver tissues (11/19 vs 18/19, P=0.019). In contrast, deletions in HBx gene were significantly higher in HCC than their non-cancerous liver tissues (16/19 vs 4/19, P<0.001). The deletion of HBx COOH-terminal was detected in 14 HCC tissues. A specific integration of HBx at 17p13 locus was also found in 8 of 16 HCC, and all of them also exhibited full-length HBx deletions. Integrated or integrated coexistence with replicated pattern was obtained in 45.5% (20/45)-56.8% (25/45) tumors and 40.9% (18/45)-52.3% (23/45) non-tumor tissues.

Conclusion: HBx deletion, especially the COOH-terminal deletion of HBx is a frequent event in HBV-associated HCC tissues in China. HBV integration had also taken place in partial HCC tissues. This supporting the hypothesis that deletion and probably integrated forms of the HBx gene may be implicated in liver carcinogenesis.

Figure 1

PCR amplified products of HBx-DNA. The fragment sizes of PCR products range from 150 bp to 800 bp. M: Marker; T: Tumor; N: Non-tumor.

Figure 2

HBx sequencing in cancer and non-cancerous tissues from 20 HBV-associated HCC patients. The amino acid sequences of HBV adr and adw subtypes are shown at the top or the bottom. Identical amino acid residues are represented by dots. The underlined amino acids were deduced from cellular flanking sequences. The frequencies of HBx point mutations were significantly lower in HCC than in the corresponding non-cancerous liver tissues (11/19 vs 18/19, P = 0.019). T: Tumor; N: Non-tumor. “…”: Represent the corresponding PCR amplificated base sequences.

Figure 3

(A) GGA→GGT sense mutations at 67aa and (B) ATT→ACT mis-sense mutations at 127aa in non-cancerous liver tissues. C: Control; N: Non-tumor.

Figure 4

Sequencing of HBx deletion mutation in HCC.

Figure 5

Sequencing of a specific integrated HBV at GA-rich region of 17p13 locus from HCC tissues of no. 2 patient. A: Tumor; B: Non-tumor.

Figure 6

A: Detection of integrated HBx fragments in HCC tissues by Southern blot analysis; B: HBc and HBx fragment amplified by PCR. M: Marker; T: Tumor; N: Non-tumor.