| Background Hepatocellular carcinoma (HCC) is the second most common fatal cancer in Southeast Asia and China. Epidemiological studies have convincingly shown that HCC is closely associated with chronic HBV infection. The incidence of HCC in chronically HBV-infected individuals is approximately 100-fold higher than in the uninfected population and the lifetime HCC risk of males infected with HBV at birth is estimated to approach 40%. Most cases of HCC occur after many years of chronic hepatitis which can provide the mitogenic and mutagenic environment to precipitate random genetic alterations and lead to the development of HCC. A study from Taiwan further demonstrated a decline in the incidence of HCC in children after implementation of a universal hepatitis B vaccination programme. HBV is a partial double-stranded DNA virus with a 3.2 kb genome containing four known open reading frames, namely, S, C, P, and X. The HBV X gene (HBx) is the smallest one, with a length of 465 bp, encoding a 154-amino acid activator protein with a molecular weight of 17 kDa. Among these four genes, HBx is most frequently integrated into the host genome, the HBx protein may function as a transactivator of various cellular genes associated with growth control and is often considered as an important factor in HBV-related hepatocarcinogenesis. The expression of HBx in the majority of HCC samples strongly suggests that it may play a critical role in regulating the proto-oncoproteins necessary for tumorigenicity and the anti-apoptotic ability of HCC. Furthermore, during the natural history of HBV infection, mutations tend to accumulate in the HBV genome。Some types of HBx changes have been detected preferentially in patientswith advanced liver disease. Firstly, some point mutations in HBx, in particular the double substitution K130M and V 131 I, are more frequent in the sera of patients with cirrhosis and/or HCC than in patients with mild liver disease. Secondly, an insert mutation at position 204 (Insert AGGCCC) always accompanied with point 260 (G→A) and 264 (G/C/T→A) was found to be the most frequent mutant pattern in either tissue (40.0%) or serum (70.7%) samples from HCC patients. Thirdly, disruptions in the HBx leading to the synthesis of C-terminally truncated proteins have been more frequently detected in tumour livers than in non-tumour ones. These C-terminally truncated HBx protein had lost their anti-proliferative effect and cooperated with ras and myc oncogenes in cell transformation. Although it is known that HCC develops many years after HBV infection, the exact molecular mechanisms of HBV in HCC have not yet been well understood.Objective (1) To investigate the distribution ofHBV genotype of hepatocellular carcinoma (HCC) patients from southern China such as Hunan, Hubei and Jiangxi provinces. (2) To investigate whether there are particular HBx gene mutations associated with HCC patients from southern China. (3) To construct the eukaryotic expression vector of HBx-d382 and HBx-d431 genes for exploring the relationship between HBx deletion mutant and hepatocellular carcinoma. (4) To express HBx-d382 and HBx-d431 genes of the eukaryotic expression vector and study the effect of HBx-d382 and HBx-d431 proteins on the biological character of QSG7701 hepatocytes. (5) To study the tumorigenicity of pcDNA3/HBx-d382 and pcDNA3/HBx-d431 and explore biological effects of truncated HBx on QSG7701 cell line.Methods (1) HBV genotypes were determined with nested-PCR by using type-specific primers in 63 HCC tumour tissues, control samples were done in 60 HBV carriers. (2) The HBx gene was examined in 51 paraffin-embedded tumor tissue samples from patients with HCC and 25 serum samples from the HBV carrier by nested polymerase chain reaction (PCR), single-stranded conformational polymorphism(SSCP) and heteroduplex analysis(HA). HBx genes with potentially important mutations from tumor tissue samples were cloned, sequenced and aligned with the published HBx gene sequence. HBV genotypes in tumor tissue samples were analyzed by nested PCR. (3) HBx-d382 and HBx-d431 genes with Kpn I and ApaI endoenzyme sites were obtained by using PCR for which pGM-T/HBx-d382 and pGM-T/HBx-d431 plasmids were used as template DNA. Vector pcDNA3, HBx-d382 and HBx-d431 were digested with double enzymes. Vector and HBx gene fragments were connected by using T4 DNA ligase and transferred into E.coli DH5a, recombinant plasmid sequence were verified by Kpn I and Apa I endoenzyme and auto-sequencing assay and named as pcDNA3/H x-d382 and pcDNA3/HBx-d431. (4) Gene transfection mediated by the lipofectamine was used to introduce the eukaryotic expression vector of HBx-d382, HBx-d431 and HBx-2215 genes into QSG7701 cell lines, respectively. The selective medium containing G418 was used to select the cell clones which express the HBx protein constantly. (5) HE staining was used to observe the morphous of cells in each group. The biological characteristics of QSG7701 cell lines in each group were analyzed by MTT assay, soft agar colony formation assay, flow cytometry(FCM) and xenograft in nude mice.Results (1) Of the 63 HCC patients, the genotypes ofHBV genomic DNA of 35 HCC patients were successfully determined by PCR with type-specific primers. 12(34.3%) were genotype B, 17(48.6%) were genotype C, 4(11.4%) were genotype B+C and 2(5.7%) were genotype B+D. Of the 60 HBV carriers, 28(46.7%) were genotype B, 24(40.0%) were genotype C, 7(11.6%) were mixed genotype B +C and 1(1.7%) were mixed genotype B +D. (2) The deletion mutation and point mutation were found in the HBx gene of HCC tumour tissue, and there are some difference between the HBx gene mutation in the genotype B and that in the genotype C. Analyses of HBx gene polymorphism showed that 31.3% of HBx gene fragments in tumor tissue samples had a special pattern. A common deletion at nt 382-400 of the HBx gene accompanied by 29 point mutations was detected in four randomly selected tumor tissue samples with this pattern which caused a frame-shift in the HBx open reading frame with a new stop codon at nt 1818, resulting in a generation of HBx polypeptide chain truncated at the C end in these cases. Among the four randomly selected samples, three were HBV genotype B, and one was not detected by our present assay. In another tumor tissue sample, amplification of the full-length HBx gene yielded a shorter fragment than expected. Sequencing of this fragment revealed a 264 bp deletion between nt 1577 and 1840 of the HBV gene. Moreover, a deletion at nt 431-445 of the HBx gene was detected in a tumor tissue sample which was HBV genotype B. (3) We have constructed the eukaryotic expression vector of HBx-d382 and HBx-d431 that carry integrated HBx-d382 or HBx-d431 gene fragment. (4) The cell clones which expressed the HBx-d382, HBx-d431 and HBx-2215 protein were constantly obtained by the selective medium containing G418. (5) QSG7701 cells grew faster in pcDNA3/HBx-d382, pcDNA3/HBx-d431 and pcDNA3/HBx-2215 groups, The colony formation rate was significantly higher in pcDNA3/HBx-d382, pcDNA3/HBx-d431 and pcDNA3/HBx-2215 groups than in pcDNA3 group [(18.27±1.35)%, (16.37±1.37)% and (15.40±1.44)% vs.(1.72±0.25)%, p＜0.05]. Compared with pcDNA3 group, pcDNA3/HBx-d382 and pcDNA3/HBx-2215 promoted more cells from G1 phase into S phase in cell cycle (29.4% vs. 32.8% and 35.0% in S phase), the apoptosis rate of pcDNA3/HBx-d431 QSG7701 cell line was singnificantly lower (13.1% vs. 4.5%). pcDNA3/HBx-d382-/QSG7701 and pcDNA3/HBx-431/QSG7701 cell lines could form tumor in nude mice in 2 weeks, which was earlier than pcDNA3/HBx-2215-/QSG7701 cell line (5 weeks).The size of tumor in nude mice was obviously larger in pcDNA3/HBx-d382 and pcDNA3/HBx-d431 groups than in pcDNA3/HBx-2215 and pcDNA3 groups.Conclusion (1) HBV genotype B, C, mixed B+C and mixed B+D exist in HCC tumour tissue samples, and genotype C is the major genotype. (2) HBx gene mutation occurs frequently in HCC sample, the mutation in the genotype B were difference from that in the genotype C. The deletion at nt 382-400 of the HBx gene might play a role in carcinogenesis of HCC in southern China. (3) pcDNA3/HBx-d382 and pcDNA3/HBx-d431 eukaryotic expression vectors are helpful in studying the role of HBx-d382 and HBx-d431 genes and proteins in carcinogenesis of hepatocellular carcinoma, and provide gene material for studying the biological functions of HBx-d382 and HBx-d431 genes and proteins.(4) we have successfully estabilished QSG7701 cell lines stably transfected HBx-d382, HBx-d431 and HBx-2215 genes, which might be valuable cell models for studying HBx-d382 and HBx-d431 genes and proteins in the development of HBV-related hepatocellular carcinoma in vitro. (5) Compared with HBx-2215 (HBx of HepG2.2.15), HBx-d382 and HBx-d431 can promote the proliferation of QSG7701 cells and have tumorigenic ability. HBx deletion mutants might play a role in the development of HCC through modifying the biological functions of HBx.
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