Hepatitis B Virus X Gene Research Articles

Hepatitis B virus X gene impacts on the innate immunity and immune-tolerant phase in chronic hepatitis B virus infection.

The immunologic features involved in the immune-tolerant phase of chronic hepatitis B (CHB) virus (HBV) infection are unclear. The hepatitis B virus X (HBx) protein disrupts IFN-β induction by downregulating MAVS and may destroy subsequent HBV-specific adaptive immunity. We aimed to analyse the impacts of genetic variability of HBx in CHB patients on the immune-tolerant phase during long-term follow-up. Children with CHB in the immune-tolerant phase were recruited and followed longitudinally. HBx gene sequencing of infecting HBV strains was performed, and the effects of HBx mutations on the immune-tolerant phase were assessed. Restoration of the host immune response to end the immune-tolerant phase was investigated by immunoblotting, immunostaining, ELISA and reporter assays of MAVS/IFN-β signalling in liver cell lines, patient liver tissues and the HBV plasmid replication system. A total of 173 children (median age, 6.92 years) were recruited. Patients carrying HBx R87G, I127V and R87G + I127V double mutations exhibited higher cumulative incidences of immune-tolerant phase breakthrough (p=.011, p=.006 and p=.017 respectively). Cells transfected with HBx R87G and I127V mutants and pHBV1.3-B6.3 replicons containing the HBx R87G and I127V mutations exhibited statistically increased levels of IFN-β, especially under poly(I:C) stimulation or Flag-MAVS cotransfection. HA-HBx wild-type interacted with Flag-MAVS and enhanced its ubiquitination, but this ability was diminished in the R87G and I127V mutants. HBx suppresses IFN-β induction. R87G and I127V mutation restored IFN-β production by preventing MAVS degradation, contributing to curtailing the HBV immune-tolerant phase in CHB patients.

Role of histone demethylase KDM6B in HBx-mediated podocyte-macrophage transdifferentiation

Objective: To explore the expression of lysine (k)-specific demethylase 6B (KDM6B) in the renal tissues of hepatitis B virus-associated glomerulonephritis (HBV-GN) patients and human podocytes transfected with hepatitis B virus X (HBx) gene, and its role in HBx-mediated podocyte-macrophage transdifferentiation (PMT). Methods: Forty-eight patients diagnosed as HBV-GN by renal biopsy from 2013 to 2018 at the Shanghai Jiaotong University Affiliated First People's Hospital were included in this study. Thirty patients with primary glomerulonephritis (PGN) and fifteen patients with renal tumor were chosen as control group. The expression of KDM6B and macrophage marker F4/80 in renal tissues of HBV-GN patients was observed by immunofluorescence and immunohistochemistry. The association between kidney KDM6B levels and clinical features of HBV-GN patients was analyzed. The expression of KDM6B, F4/80, major histocompatibility complex (MHC)-Ⅱ and CD40 in the podocytes was detected by Western blotting. The contents of interferon-γ (IFN-γ) and interleukin-6 (IL-6) in the supernatant were determined by enzyme-linked immunosorbent assay (ELISA). The small interfering RNA of KDM6B (KDM6B siRNA) was used to silence the expression of KDM6B and the protein levels of KDM6B, F4/80 and tri-methylation of lysine 27 of histone H3 (H3K27me3) induced by HBx gene transfection were detected by Western blotting. Results: Renal KDM6B expression was significantly increased in HBV-GN patients compared to normal control (0.022±0.004 vs 0.006±0.002, P=0.006). There was no significant difference in the positive rate of KDM6B among different pathological types of HBV-GN (P=0.139). Moreover, co-expression of KDM6B and F4/80 could be observed in the podocytes of HBV-GN patients. Patients with estimated glomerular filtration (eGFR)<60 ml·min-1·(1.73 m2)-1or proteinuria ≥ 3.5 g/day had a significantly higher renal KDM6B expression compared to control groups (all P<0.05). In addition, the expression of KDM6B, F4/80, MHC-II and CD40 was significantly up-regulated in the podocytes transfected with HBx gene (all P<0.05). The content of IFN-γ and IL-6 in the supernatant was significantly increased (all P<0.05). After gene silencing of KDM6B, the expression of F4/80 induced by HBx in the podocytes was significantly down-regulated, while the level of H3K27me3 was significantly increased (both P<0.05). Conclusions: HBx could induce KDM6B expression in podocytes and initiate PMT, thereby involving in the dysfunction of immune microenviroment in the renal tissues of HBV-GN.

Effect of hepatitis B virus X gene integration on expression of zinc finger protein ZBTB20 in chronic hepatitis B patients complicated with hepatocellular carcinoma

Objective To investigate the effect of hepatitis B virus (HBV) X gene integration on expression of zinc finger protein ZBTB20 in chronic hepatitis B (CHB) patients complicated with hepatocellular carcinoma (HCC). Methods Eighteen CHB patients complicated with HCC who underwent surgical treatment in Taizhou Enze Medical Center Enze Hospital and Taizhou Central Hospital from July 2015 to June 2017 were enrolled. Samples of carcinoma tissue, para-carcinoma tissue and corresponding normal liver tissue were collected from each case. DNA was extracted from three kinds of tissue samples. HBV-Alu-polymerase chain reaction (PCR) was used to amplify the integrated HBVX fragments and their bilateral flanking sequences in human genomic DNA. The integrated HBV fragments were determined by PCR products sequencing. Protein was extracted from three kinds of tissue samples.The level of expression of ZBTB20 was detected by protein imprinting. Statistical analysis was performed using t test, analysis of variance, and χ2 test. Results Among the 18 CHB patients complicated with HCC, integration of HBVX gene was found in 13 carcinoma tissue samples, 16 para-carcinoma tissue samples and 9 corresponding normal liver tissue samples. The difference was statistically significant (χ2=6.353, P=0.037). Of the 18 patients, the protein expressions of ZBTB20 in carcinoma tissue, para-carcinoma tissue and corresponding normal tissue were (50.14±11.25)%, (40.71±7.17)% and (39.06±5.17)%, respectively, which was statistically different (F=9.420, P<0.01). HBVX gene integration was detected at ZBTB20 locus in five patients. The expression levels of ZBTB20 in patients with HBVX gene integration at this locus in carcinoma tissue, para-carcinoma tissue and corresponding, normal liver tissue were all significantly lower than those in patients without HBVX gene integration (carcinoma tissue (37.37±10.30)% vs (55.06±7.06)%, para-carcinoma tissue (32.06±2.61)% vs (44.04±5.24)%, corresponding normal tissue (34.66±5.59)% vs (40.76±4.04)%, t=4.205, 4.821 and 2.589, respectively, all P<0.05). Conclusions Incidence of HBVX integration in para-carcinoma tissue is higher than that in carcinoma tissue in CHB patients complicated with HCC.The expression level of ZBTB20 in carcinoma tissue is higher than that in para-carcinoma tissue. Integration of HBVX gene at ZBTB20 locus may decreases the expression of ZBTB20. Key words: Hepatitis B virus; Carcinoma, hepatocellular; Gene integration; Zinc finger protein ZBTB20

Abstract 1814: Carboxyl-terminal truncated HBx represses lncRNA-LOC to promote cell metastasis

Abstract Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancy in the world. The poor prognosis of HCC is partly due to a high incidence rate of extrahepatic metastases. Several studies have shown that the relative risk of HCC among hepatitis B virus (HBV) carrier is higher than non-carrier group. Among the four proteins translated by HBV, X-gene (HBx) has been correlated with oncogenesis of HCC. Meanwhile, it is well known that random HBV genome integration can cause truncation of the HBV genome, especially on the HBx gene locus at the C-terminus (designated as Ct-HBx). The discovery of long non-coding RNA (lncRNA) transcripts has dramatically expanded our understanding of cell biology, especially in cancer. However, the underlying molecular mechanisms of lncRNAs that mediate the metastatic cascade and cancer progression remain largely unclear in liver cancer. In this study, lncRNA-LOC is significantly downregulated in HCC and negatively correlated with overall survival in HCC patients. LncRNA-LOC is negatively regulated by Ct-HBx in vitro and in vivo (HCC specimens). Two novel transcripts of LncRNA-LOC in HepG2 and Huh7 cell lines were identified by 5’ and 3’ rapid amplification of cDNA ends assays, respectively. Overexpression of lncRNA-LOC induced marked inhibition of cell migration, invasion and cell proliferation in vitro. On the other hand, these phenotypes were reversed upon knockdown of lncRNA-LOC in cells. Mechanically, levels of p-STAT3 (Tyr705 and Ser727), p-c-Jun (Ser63) and Snail were decreased in lncRNA-LOC-overexpressing cells, compared to control cells. Our data collectively highlight that novel regulatory associations among lncRNA-LOC, Ct-HBx, p-STAT3, p-c-Jun and Snail are an important determinant of metastasis in HCC cells and support the potential utility of lncRNA-LOC as a therapeutic strategy for HCC. Citation Format: Yang Hsiang Lin, Chau-Ting Yeh, Kwang-Huei Lin. Carboxyl-terminal truncated HBx represses lncRNA-LOC to promote cell metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1814.

HBxAg suppresses cell apoptosis and promotes the secretion of placental hormones in human placental trophoblasts via activation of the EGFR/Akt pathway.

The aim of this study is to investigate the role of Hepatitis B virus x (HBx) in the growth and secretion of human placental trophoblasts. Firstly, placenta tissues were collected from pregnant HBV carriers with various viral loads. The results of immunohistochemical technique showed that the HBx protein and pEGFR protein levels were both markedly increased with the viral load elevation. Then, a placental trophoblast cell strain (JEG-3-HBx), which stably expressed HBx mRNA and protein, was established with the pcDNA-HBx transfection followed by the G418 selection. The JEG-3-HBx strain displayed distinct activation of the EGFR/AKT pathway, a lower level of cell apoptosis, and higher secretion levels of placental hormones, including human chorionic gonadotropin (hCG), progesterone, estrogen and β-endorphin. Subsequently, HBx siRNA was used to silence the HBx gene in the JEG-3-HBx strain. Our data showed that the HBx siRNA transfection markedly suppressed the activation of the EGFR/AKT pathway, promoted cell apoptosis, and reduced the secretion of the placental hormones. Finally, EGF was applied to simulate the JEG-3-HBx strain with or without the HBx siRNA transfection. EGF treatment counteracted the reduction of cell apoptosis and the suppression of hormone secretion caused by HBx siRNA in the cell strain. In conclusion, the pEGFR protein was robustly upregulated in HBx-infected human placenta tissues and trophoblast cells. HBx reduces cell apoptosis and promotes the secretion of placental hormones in human placental trophoblast cells via activation of the EGFR/Akt pathway.

HBxAg suppresses apoptosis of human placental trophoblastic cell lines via activation of the PI3K/Akt pathway.

The aim of this study is to investigate the effect of hepatitis B virus X (HBx) protein on the apoptosis of placental trophoblastic cells and its potential mechanism. A pcDNA3.1 expression vector of HBx gene was built and transfected into JEG-3 and HTR-8 human placental trophoblastic cell lines, respectively. After transfection for 48 h, RT-PCR and immunofluorescence analyses showed that HBx mRNA and protein expression was detected in JEG-3 and HTR-8 cells. Flow cytometry revealed that early apoptosis of JEG-3 and HTR-8 cells was reduced by pcDNA-HBx transfection. Immunofluorescence and Western blotting showed that PI3K and p-Akt were significantly upregulated in HTR-8 cells. HBx ectopic expression did not change the viability of JEG-3 and HTR-8 cells when the PI3K/Akt pathway was blocked by its specific inhibitor LY294002. Moreover, the pcDNA-PI3K expression vector and pcDNA-HBx were transfected individually or co-transfected into the cells. The results showed that pcDNA-PI3K/pcDNA-HBx co-transfection promoted the expression of PI3K protein compared with the pcDNA-PI3K transfection group but did not increase the expression of HBx protein compared with pcDNA-HBx transfection group. In conclusion, HBx gene can be transferred into JEG-3 and HTR-8 human placental trophoblastic cell lines and cause inhibition of cell apoptosis. Its effect of apoptosis inhibition is related to the activation of the PI3K/Akt signaling pathway.

Genomic change in hepatitis B virus associated with development of hepatocellular carcinoma.

To determine the genomic changes in hepatitis B virus (HBV) and evaluate their role in the development of hepatocellular carcinoma (HCC) in patients chronically infected with genotype C HBV. Two hundred and forty chronic hepatitis B (CHB) patients were subjected and followed for a median of 105 mo. HCC was diagnosed in accordance with AASLD guidelines. The whole X, S, basal core promoter (BCP), and precore regions of HBV were sequenced using the direct sequencing method. All of the subjects were infected with genotype C HBV. Out of 240 CHB patients, 25 (10%) had C1653T and 33 (14%) had T1753V mutation in X region; 157 (65%) had A1762T/G1764A mutations in BCP region, 50 (21%) had G1896A mutation in precore region and 67 (28%) had pre-S deletions. HCC occurred in 6 patients (3%). The prevalence of T1753V mutation was significantly higher in patients who developed HCC than in those without HCC. The cumulative occurrence rates of HCC were 5% and 19% at 10 and 15 years, respectively, in patients with T1753V mutant, which were significantly higher than 1% and 1% in those with wild type HBV (P < 0.001). The presence of T1753V mutation in HBV X-gene significantly increases the risk of HCC development in patients chronically infected with genotype C HBV.

MicroRNA expression profiles of LO2 cells expressing the wild-type and mutant HBx gene

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Although numerous studies have suggested the potentially oncogenic roles of wild‑type or mutant hepatitis B virus X (HBx) protein in hepatocarcinogenesis, their exact mechanism remains unclear. Increasing evidence suggests that microRNAs (miRNAs) play essential roles in embryogenesis, cell differentiation and carcinogenesis. This study aimed to investigate the effect of HBx on the miRNA expression profile of LO2 cells. We established the LO2 cell line transfected with recombinant plasmid pcDNA3.0/HBx-d382, pcDNA/HBx and plasmid pcDNA3.0 using Lipofectamine™ 2000, which was confirmed by reverse transcription‑polymerase chain reaction (RT-PCR) and western blotting. We then demonstrated the miRNA expression profiles in the stably transfected LO2 cells using a mammalian miRNA microarray containing whole‑human mature and precursor miRNA sequences. The results were confirmed by real-time quantitative PCR (qPCR). RT-PCR and western blot analysis showed that a stably HBx‑transfected LO2 cell line had been successfully established. According to the microarray, compared to LO2/pcDNA3.0 cells, 6 miRNAs were shown to have higher expression and 5 were shown to have decreased expression in LO2/HBx-d382 cells, while 4 up- and 12 downregulated miRNAs were observed in LO2/HBx cells. There were 8 different expression patterns of miRNAs between LO2/HBx and LO2/HBx-d382 cells. All the chip results were consistent with the real‑time PCR data. Consequently, the HBx gene may influence the miRNA expression profile of LO2 cells. Thus, it may be helpful to further investigate the role of HBx in hepatocarcinogenesis and clarify the underlying molecular mechanisms involved.

Molecular characterization of hepatitis B virus X gene in chronic hepatitis B patients

BackgroundHBV-X protein is associated with the pathogenesis of HBV related diseases, specially in hepatocellular carcinomas of chronic patients. Genetic variability of the X gene includes genotypic specific variations and mutations emerging during chronic infection. Its coding sequence overlaps important regions for virus replication, including the basal core promoter. Differences in the X gene may have implications in biological functions of the protein and thus, affect the evolution of the disease. There are controversial results about the consequences of mutations in this region and their relationship with pathogenesis. The purpose of this work was to describe the diversity of HBV-X gene in chronic hepatitis patients infected with different genotypes, according to liver disease.MethodsHBV-X gene was sequenced from chronic hepatitis B patient samples, analyzed by phylogeny and genotyped. Nucleotide and aminoacid diversity was determined calculating intragenetic distances. Mutations at 127, 130 and 131 aminoacids were considered in relation to liver disease.ResultsThe most prevalent genotype detected in this cohort was F (F1 and F4), followed by D and A. Most of the samples corresponding to genotypes A and F1 were HBeAg(+) and for genotypes D and F4, HBeAg(−) samples were represented in a higher percentage. Intragenetic distance values were higher in HBeAg(−) than in positive samples for all genotypes, and lower in overlapped regions, compared to single codification ones. Nucleotide and aminoacid diversities were higher in HBeAg(−), than in HBeAg(+) samples.ConclusionsIndependently of the infecting genotypes, mutations at any of 127, 130 and/or 131 aminoacid positions and HBeAg(−) status were associated with mild liver disease in this cohort.

Analysis of hepatitis B virus X gene (HBx) mutants in tissues of patients suffered from hepatocellular carcinoma in China

Hepatitis B virus (HBV) X (HBx) gene multi-site mutations are a frequent event in the clinical hepatocellular carcinoma (HCC) tissues. It has been reported that the mutation of the HBx plays a crucial role in the development of HBV-related HCC. To identify the novel mutations of HBx in the HCC tissues, we examined and analyzed the sequences of HBx gene in 60 cases of HCC tumor tissues and paratumor tissues from China by polymerase chain reaction (PCR). The mutation patterns of HBx were analyzed by comparing the tumor tissues with non-tumor tissues. The data showed that 44 cases of tissues out of 60 patients were HBV-positive. Our results showed that the mutations at amino acid 30, 88, 144 from tumor samples and at amino acid 31, 43, 87, 94 from non-tumor samples were highly frequent events. Interestingly, we found that a novel type of HBx linked-mutations, such as at aa L30F/S144A, was 29.5% (13/44) positive in the tumor tissues. However, the role of HBx gene mutations at aa L30F/S144A relative to wild type HBx gene is unclear in hepatocarcinogenesis. The novel HBx linked-mutations may be significant in the development of HCC.

Could HBx Protein Expression Affect Signal Pathway Inhibition by Gefitinib or Selumetinib, a MEK Inhibitor, in Hepatocellular Carcinoma Cell Lines?

Hepatitis B virus X (HBx) protein has been known to play an important role in development of hepatocellular carcinoma (HCC). The aim of this study is to find out whether HBx protein expression affects antiproliferative effect of an epidermal growth factor receptor-tyrosine kinase (EGFR-TK) inhibitor and a MEK inhibitor in HepG2 and Huh-7 cell lines. We established HepG2 and Huh-7 cells transfected stably with HBx gene. HBx protein expression increased pERK and pAkt expression as well as β-catenin activity in both cells. Gefitinib (EGFR-TK inhibitor) inhibited pERK and pAkt expression and β-catenin activity in both cells. Selumetinib (MEK inhibitor) reduced pERK level and β-catenin activity but pAkt expression was rather elevated by selumetinib in these cells. Reduction of pERK levels was much stronger with selumetinib than gefitinib in both cells. The antiproliferative efficacy of selumetinib was more potent than that of gefitinib. However, the antiproliferative effect of gefitinib, as well as selumetinib, was not different between cell lines with or without HBx expression. Signal pathway activation by HBx might not be strong enough to attenuate the antiproliferative effect of EGFR-TK inhibitor. Future experiments are needed to understand the role of HBx protein expression in HCC treatment using molecular targeting agent.

(145) Prevalence of hepatitis B virus, hepatitis C virus, and HIV in patients of chronic liver disease in darfur

Backgroundl Aims: Chronic infection with hepatitis B virus is a high-risk factor for hepatocellular carcinoma in humans. The HBV X-protein, a multi-functional viral regulator, has been suspected to play a positive role in hepatocarcinogenesis, as demonstrated by the high incidence of hepatocellular carcinoma in HBx-expressing transgenic mice, although it is still controversial. The aim of this study was to generate transgenic mice expressing the HBV X-gene under authentic promoter control and to test whether the gene products can cause hepatic tumors.Methods: Three transgenic mouse lines were generated by microinjecting the X-gene construct into hybrid (C57BL/6×DBA) eggs. Gene expression was tested by protein and mRNA analyses. During an observation period of 18 months, mice were sacrificed and organs subjected to histologic examinations.Results: Grossly defined hepatocellular carcinomas reproducibly were observed in mice expressing the X-protein, which were investigated through six generations from the age of 11 to 18 months. Among 14 transgenic mice investigated from the age of 11 to 18 months, 12 were found to have hepatocellular carcinoma, grossly or microscopically. The lesion of the hepatocellular carcinoma disclosed a significant increase in the proliferating cell nuclear antigen in the nuclei.Conclusion: The incidence of hepatocellular carcinoma (86%) in our HBV X transgenic mice may be highly significant, since, except for one case, HBV X-gene transgenic mice produced in other laboratories did not develop liver tumor or any other pathologic phenomena.

An improved experimental model for studying vertical transmission of hepatitis B virus via human spermatozoa

Study of the mechanism of transmission of hepatitis B virus is important for public health. An improved experimental model is described for studying vertical transmission of hepatitis B virus (HBV) via human spermatozoa. Recombinant plasmid pIRES2-EGFP-HBx which would express enhanced green fluorescent protein (EGFP) used as a marker for the expression of hepatitis B virus X (HBx) gene was constructed successfully and confirmed by PCR, EcoR I and Sal I digestion, and DNA sequencing. After exposure to the plasmid, human spermatozoa were used to fertilize zona-free hamster ova in vitro. Two-cell embryos were collected and classified into group A with green fluorescence and group B without green fluorescence under fluorescence microscope. The results showed that HBx DNA positive bands were detected in the embryos with green fluorescence (PCR and RT-PCR) and positive controls (PCR) indicating presence and expression of HBx gene. In contrast, HBx gene expression was not detected in the embryos without green fluorescence and negative controls (PCR and RT-PCR). This improved experimental model is more efficient, accurate and reliable for studying further perinatal transmission of HBV or other viruses causing chronic human disease possibly via the male germ line.

Identification of a natural mutant of HBV X protein truncated 27 amino acids at the COOH terminal and its effect on liver cell proliferation

To identify mutants of the hepatitis B virus (HBV) X (HBx) gene and investigate the effect of the natural mutant on liver cell proliferation. 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. 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-XDelta127 cells was enhanced by the BrdU incorporation assay and flow cytometry analysis. Luciferase reporter gene assay showed that the transcriptional activities of NF-kappaB, 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. Our findings suggest that the natural HBx mutant truncated 27 amino acids at the COOH terminal promotes cell proliferation.

HBV X-gene: A new serum marker for anti-HBV therapy monitoring

Objective To address HBV serum nucleic acid markers for stages without apparent replication. Methods DNA and RNA sequence segments from the X, C and pre C/C regions produced successively during replication were used as targets for quantitative PCR and RT/PCR. Results The assays confirmed the preferential formation of intermediates blocked at early stages. They persisted as the only detectable type of serum HBV DNA even after one year of therapy. At reentry into viral replication due to emergence of drug resistant mutants, lamivudine resistance produced exclusively incomplete DNA minus strands, whereas the wild type virus immediately synthesized complete DNA minus strands. Conclusion PCR assays used for monitoring complete suppression of HBV replication must target the X gene region.

Impact of hepatitis B virus (HBV) X gene integration in liver tissue on hepatocellular carcinoma development in serologically HBV-negative chronic hepatitis C patients

We analyzed hepatitis B virus (HBV) X gene integration in hepatocytes of HBV-negative, chronic hepatitis C (CH-C) patients with mild fibrosis, and prospectively followed these patients for the development of hepatocellular carcinoma (HCC). The study included 39 HBV-negative CH-C patients with mild fibrosis. HBV-X integration was determined by Alu-PCR analysis of liver specimens obtained by fine-needle biopsy. Integration of HBV-X gene sequence into liver genome occurred in 9 of the 39 patients. Six of the 39 patients developed HCC during the 12-year follow-up period. No significant difference was found in the incidence of HCC between patients with and without HBV-X integration. However, the two patients with HBV-X integration who developed HCC did not have cirrhosis at the time when HCC was diagnosed, whereas the four patients without HBV-X integration who developed HCC did have cirrhosis. Our findings suggest that HBV-X integration detected at the mild fibrosis stage might not indicate a high risk for HCC. HBV-X integration may be associated with HCC development in the absence of cirrhosis. However, we did not find evidence that HBV-X integration directly plays a role in hepatocarcinogenesis in CH-C patients. Further studies will be needed to clarify this point.

Hepatitis B virus X protein induces apoptosis and cell cycle deregulation through interfering with DNA repair and checkpoint responses

To investigate the effects of hepatitis B virus X (HBx) gene on apoptosis and cell cycle in hepatocyte line HL-7702 and to discuss the possible mechanisms in the pathway. The recombinant plasmid pcDNA3-X and vector pcDNA3 were transfected into HL-7702 cells and selected by G418 to construct two new cell lines, which were named HL-7702-HBx and HL-7702-con, respectively. Reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis were used to confirm that HBx gene was expressed steadily in the HL-7702-HBx cells. Then apoptosis and cell cycle of the two cells were detected by DNA ladder, flow cytometric analysis, and electronic microscope observation. Apoptosis and cell cycle gene expressions in the two cells were subsequently evaluated by using gene arrays. Some of results were further confirmed by real-time PCR and western blot analysis. RT-PCR and the western blot analysis showed that HL-7702-HBx expressed the HBx gene steadily. Comparedwith the HL-7702-con cells, there was increased apoptosis and accumulation of the S phase in the HL-7702-HBx cells. The gene array analysis indicated that some DNA repair genes (XRCC1, DDB1, etc.) and DNA damage checkpoint-related genes (Cdc47, RAD17, etc.) played roles in the HBx-mediated imbalance of apoptosis and cell cycle. Both cDNA array analysis and real-time RT-PCR showed that mRNA of XRCC1, Cdc47 and RAD17 were upregulated by HBx. Unexpectedly, the western blot analysis revealed that HBx inhibited their protein expression. The expression of HBx in HL-7702 cells promoted apoptosis and accumulation of the S phase through the inhibition of DNA repair and checkpoints via post-transcriptional mechanisms.

Inhibition of Hepatitis B Virus Replication by in vitro Synthesized RNA

Human hepatitis B virus (HBV) is a pathogen related to the development of liver diseases including chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). However, the efficient methods to suppress HBV replication have not been developed yet. Therefore, we have used RNA interference (RNAi) as a potential tool for the suppression of HBV replication. Here, we designed a 21 nt small intefering dsRNA (siRNA) against hepatitis B virus X (HBx) RNA with 3' overhanging ends derived from T7 promoter. It has been reported that HBV X protein plays an important role in HBV gene expression and viral replication. The suppression of HBx gene expression by the 21 nt siRNA was investigated by Northern blot analysis and chloramphenicol acetyl transferase (CAT) assay. The level of HBx mRNA was decreased by siRNA in a dose-dependent manner. We also found that the 21 nt siRNA inhibited the HBV replication in hepatocellular carcinoma cell.

Development of PCR‐ELISA for the detection of hepatitis B virus x gene expression and clinical application

The presence of hepatitis B virus x (HBx) antigen/antibody is known to correlate with the well-established serological markers of ongoing viral replication in the chronic phase of HBV infection, and strongly suggests that the level and duration of HBx expression may influence the outcome of the chronic infection. In this research, we developed a polymerase chain reaction enzyme-linked immunosorbent assay (PCR-ELISA) method for the detection of HBx gene expression. We also investigated its relationship to the progress of the disease in HBV-related patients. Peripheral blood mononuclear cells (PBMCs) were purely isolated, and reverse transcription-PCR (RT-PCR) was performed for improved sensitivity. The PCR products were determined by ELISA, and we investigated the relationship of the proposed method to the clinical status of the patients. The PCR-ELISA used in this work was found to be at least 100 times more sensitive than the conventional PCR method, and even 8,000-fold diluted PCR products could be detected. The HBx concentrations significantly differed among control subjects (0.36+/-0.09, [P<0.01] and patients with chronic hepatitis (1.13+/-0.34 [P<0.01 compared to control]), liver cirrhosis (LC; 1.37+/-0.28 [P<0.01 compared to control]), and hepatocellular carcinoma (HCC; 1.48+/-0.95 [P<0.01 compared to control]). These findings suggest that monitoring of HBx could be useful for early diagnosis and prognosis in patients with chronic HBV infection, LC, and HCC.

The effect of hepatitis B virus X gene transfection on expression of human telomerase reverse transcriptase mRNA in human bile duct carcinoma cell lines

To study the effect of Hepatitis B virus X (HBx) gene transfection on expression of human telomerase reverse transcriptase (hTERT) mRNA in human bile duct carcinoma cell lines QBC939 and to elucidate the significance of cis-activation of hTERT mRNA by HBx gene on the carcinogenesis of bile duct. QBC939 were cultured in vitro and co-transfected with eukaryotic expression vector containing the HBx coding region and cloning vector containing enhanced green fluorescent protein (EGFP) coding sequence using liposome-mediated gene transduction technique. Thirty six hours after transfection, EGFP expression, the indicator of successful transfection in cells, was determined. Flow cytometry was applied to determine the transfection efficiency. Cells were harvested and total RNA was extracted with TRI(ZOL) Reagent. The expression of hTERT mRNA in QBC939 was assayed by Reverse Transcription Polymerase Chain Reaction. The expression of HBx protein in QBC939 was detected by immunocytochemistry staining and western blotting. The transfection efficiency was 29.6% for both HBx expression vector and vector control group. The expression of hTERT mRNA was significantly increased when transfected with HBx expression vector than that transfected with OPTI-MEM medium and vector only. The expression of HBx protein could only be found in the cells when transfected with HBx expression vector by immunocytochemistry staining and western blotting. HBx gene transfection may up-regulate the transcriptional expression of hTERT mRNA in bile duct carcinoma cells. The cis-activation of hTERT gene by HBx gene is primary mechanism for carcinogenesis of biliary epithelia after HBV infection.