HBx Gene Research Articles

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

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. 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. 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. 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.

Downregulation of HBx mRNA in HepG2.2.15 cells by small interfering RNA

Hepatitis B virus (HBV) infection is one of the most prevalent viral infectious diseases in humans. And it is still a challenge for the development of an effective therapy for HBV infection. Recently, the progress in RNA interference (RNAi) has shed some light on the inhibition of HBV expression and replication by RNAi specific for the various genes of the HBV genome. Some prior researches suggests that the HBV x protein (HBx) plays an important role in viral transcription, cell growth, and apoptotic cell death. In the present study, we designed three siRNAs based on the X-protein of HBV sequences and tested their effects on the expression of HBx gene following sorting of siRNA-positive cells. The interference effect was tested in 24, 48, and 72 h. HBsAg in cultured media was assayed using western blot at various days post-transfection. The amount of HBx mRNA was quantitated by Real-time reverse-transcript PCR (RT-PCR). There was a decrease in the levels of HBV mRNA and HBsAg from the the transfected cells. Among these three siRNAs, siRNA-2 was found to be the most effective at suppressing HBV gene expression.

Polymorphism Analyses of Hepatitis B Virus X Gene in Hepatocellular Carcinoma Patients from Southern China

Chronic hepatitis B virus (HBV) infection is one of the major causes of hepatocellular carcinoma (HCC), and the HBV X (HBx) gene plays a critical role in the molecular pathogenesis of HBV-related HCC. We have investigated whether there are particular HBx gene mutations associated with HCC in patients from southern China. 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 and heteroduplex analysis. The 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. 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 an 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. Sequencing of this fragment revealed a 264 bp deletion between nt 1577 and 1840 of the HBV gene. These results suggest that HBx gene mutation occurs frequently in HCC samples, and the deletion at nt 382-400 of the HBx gene might play a role in carcinogenesis of HCC in southern China.

The effect of HBx gene on the apoptosis of hepatic cells

To study the effect of HBx gene on the apoptosis of the cell lines (L02, HepG2) and the interaction between HBx and X-linked inhibitor of apoptosis protein (XIAP), the apoptosis of pcDNA3.1-HBx transiently transfected cell lines (L02, HepG2) was detected by flow cytometry and the mRNA expression of XIAP was assayed by real-time RT-PCR. Our study showed (1) the morphology of L02/pcDNA3.1-HBx was changed and the appearance of the cells mimicked that of HepG2 cells; (2) HBx gene could be detected in L02/pcDNA3.1-HBx and HepG2/ pcDNA3.1-HBx; (3) the apoptosis rate of L02/pcDNA 3.1-HBx was higher than that of L02 cells (P<0.01) and the apoptosis rate of HepG2/pcDNA3.1-HBx was lower than that of HepG2 cells (P<0.05); (4) the XIAP expression in L02 was about 3 times that in L02/pcDNA3.1-HBx cells (P<0.01), and the expression of XIAP in HepG2/pcDNA3.1-HBx was about 4 times that in HepG2 (P<0.01). It is concluded that HBx gene may promote the apoptosis of normal hepatocytes and inhibit the apoptosis of cells of hepatic carcinoma by regulating the expression of XIAP.

HBx or HCV core gene expression in HepG2 human liver cells results in a survival benefit against oxidative stress with possible implications for HCC development

Hepatitis virus replication in the liver is often accompanied by inflammation resulting in the formation of reactive oxygen species (ROS) and nitric oxide (NO) and these may induce cell death. We investigated whether the expression of HBx or HCV core protein in HepG2 cells has an influence on the sensitivity of these cells for oxidative radicals. Our previous study, using the inducible HBV model of HepAD38, revealed that oxidative-stress-related genes are upregulated by virus replication. In the present study, we examined the intracellular pro-oxidant status with dichlorofluorescein (DCF) in HepG2 cell lines transfected with HBx, HbsAg and HCV core. Baseline intracellular oxidative levels were not different in the cell lines expressing viral proteins as compared to control. However, when these cells were exposed to H 2O 2, the viral protein expressing cells, especially those expressing HBx, showed a reduced level of ROS. This suggests that HBx and HCV core transfected cells can convert H 2O 2 to less reactive compounds at a higher rate than the control cells. When HBx or HCV core expressing cells were exposed to peroxynitrite (a highly reactive product formed under physiological conditions through interaction of superoxide (O 2 − ) with NO) these cells were less sensitive to induction of cell death. In addition, these cell lines were less prone to cell death when exposed to H 2O 2 directly. In conclusion, HBx and HCV core expression in HepG2 cells leads to a survival benefit under oxidative stress which in vivo can be induced during inflammation.

The Upregulation of Expressed Proteins in HepG2 Cells Transfected by the Recombinant Plasmid‐containing HBx Gene

It is known that the hepatitis B virus X protein (HBx) plays a crucial role in the pathogenesis of HCC, but the exact functions and molecular mechanisms of HBx in HCC are not well understood. In the present study, HepG2 cell lines were cultured and transfected with pEGFP-N1 and pEGFP-N1-X. Twenty-four hours after transfection, cells were harvested and total RNA was extracted using TRIzol reagent. The expression of HBx in HepG2 cell line was assayed by real-time polymerase chain reaction and was detected by Western blotting. Moreover, proteomic analysis was performed for the HepG2-pEGFP-X cells and HepG2-pEGFP control cells. The combination of 2DE and MALDI-TOF-MS/MS revealed that SEC13L1 (SEC13-like 1 isoform b), PA28 alpha (proteasome activator REG alpha), serine-threonine kinase receptor-associated protein (STRAP) and nm23/nucleoside diphosphate kinase (NME) were upregulated in HepG2-pEGFP-X cells. STRAP is known to be a WD40 domain-containing protein, which interacts with TbetaR-I and TbetaR-II and negatively regulates TGF-beta signalling, was also found increased in human cancers. NME is known to be involved in the regulation of cancer cell progression and metastasis. These results would help the understanding of how HBx maintains tumorigenicity and progression of HCC.

Inhibition of hepatitis B virus X gene expression by 10-23 DNAzymes

The X protein (HBx) of human hepatitis B virus (HBV) is a transcriptional activator protein. The HBx protein plays an important role in viral replication in HBV infected cells and the liver diseases including hepatitis, cirrhosis and hepatocellular carcinoma (HCC). Therefore, the repression of HBx gene expression by 10-23 DNAzymes might be a good way to inhibit HBV replication and counteract HBV-related liver diseases. We designed three 10-23 DNAzymes with different substrate-recognition domains. When each of the 10-23 DNAzymes were cotransfected into human AD293 cells with HBx-EGFP expression plasmid, they could all reduce the level of HBx mRNA as well as the HBx-EGFP protein. These results suggest that the 10-23 DNAzymes might be used for gene therapy of liver diseases caused by HBV.

Antisense regulation of expression and transactivation functions of the tumorigenic HBx and c-myc genes

Earlier we have shown that the X- myc transgenic mice develop hepatocellular carcinoma (HCC) due to co-expression of c-Myc and HBx protein of hepatitis B virus [R. Lakhtakia, V. Kumar, H. Reddi, M. Mathur, S. Dattagupta, S.K. Panda, Hepatocellular carcinoma in a hepatitis B ‘x’ transgenic mouse model: a sequential pathological evaluation. J. Gastroenterol. Hepatol. 18 (2003) 80–91]. With the aim to develop therapeutic strategies for HCC, we constructed several mono- and bicistronic antisense recombinants against HBx and c-myc genes to regulate their expression as well as transactivation function in a human hepatoma cell line. A dose-dependent inhibition in the expression levels of HBx and c-Myc was observed with monocistronic constructs. Likewise, the bicistronic recombinants also blocked the expression as well as transactivation functions of cognate genes with equal efficacy. Further, expression of the constituent genes from the X- myc transgene could also be inhibited by these antisense constructs in cell culture. Thus, our study points towards clinical implications of antisense regulation of tumor-promoting genes in the management of HCC.

Hepatocellular carcinoma cells containing hepatitis B virus X protein have enhanced invasive potential conditionally

Aims To establish a sustaining hepatitis B virus X protein expressed Chang liver cell line and to explore their biological behaviours of invasive potential induced by hepatitis B virus X protein. Methods Polymerase chain reaction was used to amplify the HBx gene from the whole hepatitis B virus genome. The gene was then subcloned into the eukaryotic expression vector pcDNA3.1 to construct the pcDNA3.1-HBx plasmid. Gene transfection mediated by Lipofectamine was used to introduce the plasmid into the human liver cell line Chang, and stable expression of the HBx gene was detected. Results HBx gene was cloned from the transfected Chang liver cells by reverse transcription-polymerase chain reaction, and confirmed by electrophoresis. The stably transfected Chang cells expressing HBx with malignant characteristics were verified and compared with control cells in terms of their growth curves, clonogenicity, wound healing abilities, migration and metastasis. Conclusion The stabilising human liver cell lines Chang liver containing HBx gene expression have been established successfully. The invasive potential of Chang cells was conditionally enhanced by HBx transfection.

Hepatitis B virus X protein upregulates survivin expression in hepatoma tissues

The hepatitis B virus X protein (HBx) plays an important role in the development of hepatocellular carcinoma (HCC). The relationship was examined between HBV antigens and IAP (inhibitor of apoptosis) family in development of HCC. The expression levels of HBV antigens (HBsAg, HBcAg, and HBxAg) and members of the IAP family (survivin, XIAP, cIAP-1, and cIAP-2) were detected immunohistochemically in tissues from 34 cases of HCC and 30 cases of liver cirrhosis. The positive rate of survivin was higher than these three molecules in all three tissue types (P < 0.05). The positive rates of HBxAg and survivin were high in HCC (76.5% and 88.2%), paratumor (85.3% and 91.2%), and liver cirrhosis (100% and 93.3%) tissues, with no significant differences between the survivin- and HBxAg-positive rates (each P > 0.05). To examine the effect of HBx on survivin expression, plasmid pCMV-X (encoding the HBx gene) was transfected transiently with or without plasmid pcDNA3-sur (encoding the survivin gene) into H7402 hepatoma cells and L-O2 human normal liver cells. Cells over-expressing HBx alone showed increased apoptosis along with a dose-dependent increase in survivin levels. However, co-expression of survivin inhibited the HBx-induced apoptosis. To examine the effect of HBx on survivin in hepatoma cells without apoptosis, plasmid pCMV-X was transfected stably into human hepatoma H7402 cells and L-O2 cells. These H7402-X and L-O2-X cells showed high-level expression of both HBx and survivin, but did not show apoptosis. The addition of pSilencer 3.0-X, an RNAi vector targeting the HBx gene, reduced the expression levels of survivin protein in H7402-X cells. Collectively, these data demonstrate that HBx upregulates survivin expression in hepatoma tissues, suggesting that HBx and survivin may both be involved in carcinogenesis of HCC.

HBx M130K and V131I (T-A) mutations in HBV genotype F during a follow-up study in chronic carriers

BackgroundAround 400 million people worldwide are chronically infected with Hepatitis B virus (HBV). An estimated 10% of these chronic patients develop progressive liver damage including cirrhosis and Hepatocellular Carcinoma (HCC). The HBx gene encodes a protein of 154 amino acids which is a transactivator and has been associated with HBV pathogenesis. A change in the amino acid sequences at positions 130 and 131 in the HBV-X protein (M130K and V131I) produced by T-A point mutations at the nucleic acids level has been associated with severe liver damage and HCC in patients from China and Africa. Further, such changes have been proposed as a prognostic marker for progressive liver damage and HCC. The purpose of this study was to determine if T-A mutations are present in HBV chronic carriers with genotype F (the major genotype in Costa Rica) and further, if these mutations are associated with HBV disease progression in Costa Rica HBV patients from 1972 to 1985.ResultsSerum samples from 50 HBV positive individuals were amplified and directly sequenced, 48 belonged to genotype F, 1 from genotype D and another was classified as D or E.T-;A mutations were absent in 17 acute patients who recovered, but was present in 12 of 29 chronic carrier samples (42.8%), in one sample the T-A mutations were detected as early as 29 days after clinical onset of disease. In 17 carriers with available liver biopsies, T-;A mutations were found in 8 sera of 13 (61.5%) classified as moderate or severe, and none in 4 biopsies with mild liver damage. However, it was not possible to demonstrate a statistical association between the presence of T-A mutations and moderate/severe liver damage, using a Fischer exact test, 1 tail, p = 0.05.In 4 patients HCC was diagnosed, and 2 of them presented the T-A mutations in their sera.ConclusionT-A mutations were found in HBV genotype F in chronic carriers but not in patients who recovered from acute infection. These mutations could be developing early during infection although the possibility of infection with the mutant virus could not be excluded.More studies are necessary to establish if the T-A mutation can be used as a prognostic marker for severity of liver disease in patients infected with HBV.

Hepatitis B virus X protein modulates the apoptosis of hepatoma cell line induced by TRAIL

The purpose of this study is to observe the effects of HBx on the apoptosis of hepatoma cells induced by TNF-related apoptosis-inducing ligand (TRAIL) and to study preliminary molecular mechanisms for its effects. In order to set up a model in vitro, BEL7402-HBx cell line, stably expressing HBx mRNA, was established by stable transfection of pcDNA-HBx, which contains HBx gene, into hepatoma cell line BEL7402. Control cell line BEL7402-cDNA3, stably transfected with pcDNA3, was set up simultaneously as a control. Trypan blue exclusion test, caspase 3 activity detection and TUNEL assay were performed to detect the apoptosis of BEL7402, BEL7402-cDNA3, BEL7402-HBx induced by TRAIL. The expression of TRAIL receptors in three groups was analyzed by Flow cytometry. In addition, phosphorothioated antisense oligonucleotide against the translation initial region of HBx gene (PS-asODNs/HBx) was used to block the expression of HBx in HepG2.2.15 cells and to further confirm the effects of HBx on TRAIL-induced apoptosis. Trypan blue exclusion test indicated that TRAIL had a dose-dependent cytotoxicity on BEL7402, BEL7402-cDNA3 and BEL7402-HBx cells. Under treatment of the same concentration of TRAIL, BEL7402-HBx had a higher apoptosis rate and a higher level of Caspase 3 activation than BEL7402 and BEL7402-cDNA3. TUENL assay showed that the apoptosis rate of BEL7402-HBx induced by 10 microg/L TRAIL was 41.4% +/- 7.2%, significantly higher than that of BEL7402 and BEL7402-cDNA3 cells. Blockade of HBx expression in Hep G2.2.15 cells partly inhibited the apoptosis induced by TRAIL. The introduction or blockade of HBx did not change the expression pattern of TRAIL receptors. The present study firstly confirms the effects of HBx on TRAIL- induced apoptosis from two different points and it is not related with the expression level of TRAIL receptors. This would be useful to further clarify the roles of imbalanced apoptosis in pathogenesis of Hepatitis B and related hepatocellular carcinoma.

Inhibition of Hepatitis B virus gene expression by single and dual small interfering RNA treatment

RNA interference (RNAi) has been successfully applied in suppression of Hepatitis B virus (HBV) replication. To circumvent the problem that mutation in HBV genome may result in resistance when siRNA is further developed as an anti-viral drug, in this study, we established a dual small interfering RNA (siRNA) expression system, which could simultaneously express two different siRNA molecules that can specifically target two genes. To test the effectiveness of this system, we applied this new approach to express simultaneously two different 21-bp hairpin siRNA duplexes that specifically attack the HBs and HBx genes of HBV, respectively, in Bel-7402 and HepG2.2.15 cells. Results indicated that dual siRNA could simultaneously inhibit the expression of HBs and HBx gene by 83.7% and 87.5%, respectively, based on luciferase assays. In addition, dual siRNA molecules were able to significantly reduce the amount of HBV core associated DNA, which is considered as an intracellular replicative intermediate, and the viral DNA in culture supernatant. Therefore, this dual siRNA system provides a more powerful tool for the study of gene function and implicates a potential application in the treatment of viral infection.

Identification of hepatitis B virus X gene mutation in Hong Kong patients with hepatocellular carcinoma

Chronic infection by hepatitis B virus (HBV) is the leading cause of hepatocellular carcinoma (HCC) in man. The viral transactivator HBV X (HBx) gene plays a critical role in the molecular pathogenesis of HBV-related HCC. The aim of this study was to investigate whether there were particular HBx mutations associated with the Chinese Hong Kong patients with HCC. We have examined HBx in 113 tumor tissue samples from patients with HCC and 48 serum samples from the same group. In addition, we also examined the expression of HBx protein and the index of apoptotic cell death in tumor tissues of HCC. The entire coding region of HBx gene from the sample was sequenced and aligned with the published HBx gene sequence. We have identified total 54 different types of mutations in HBx gene. HBx mutations occurred in a very high percentage of samples tested. Mutation of HBx was found in 95.2% and 95.3% of the tumor tissue and serum samples, respectively. Most of samples contained more than one type of the mutation. Relative risk analysis indicated that the mutations in 12 sites of tissue HBx and nine sites of serum HBx were highly associated with HCC, suggesting a potential role of these mutants in carcinogenesis. An insert mutation at position 204: Insert 204AGGCCC, was always found to co-exist with point mutations at 260 (G-->A) and 264 (G/C/T-->A). Furthermore, this particular pattern of HBx mutation was most frequently detected. Immunochemical staining of HBx protein revealed that the nuclear localization of HBx protein in hepatocytes of tumor tissues was highly associated with this particular pattern of HBx mutation. In conclusion, HBx mutation occurs frequently in HCC samples tested and a sample usually has multiple types of mutations. A special pattern of insert at 204 and point mutations at 260 and 264 was identified, and it appears to be associated with the nuclear localization of HBx protein. The development of multiple types of mutations in a given sample may contribute to the process of multiple steps in hepatocarcinogenesis.

Effects of hepatitis B virus X protein on human telomerase reverse transcriptase expression and activity in hepatoma cells

In subjects with hepatitis B, carcinogenesis has been associated with the hepatitis B virus (HBV) X protein (HBX) and human telomerase reverse transcriptase (hTERT). In the experiments reported here, we used immunohistochemical methods to study the expression of hTERT and HBV antigens (HBsAg, HBcAg and HBxAg) in 34 cases of HCC and corresponding paratumor tissues, 30 cases of liver cirrhosis, and 6 normal livers. To examine the effect of HBX on hTERT expression and activity in hepatoma cells, we transiently and stably transfected the pCMV-X plasmid cloned HBx gene into H7402 hepatoma cells, then measured the expression of c-Myc and hTERT in these cells with the use of Western-blot analysis. Telomerase activity was detected with the use of the telomerase repeat amplification protocol (TRAP) in transiently and stably transfected cells. We found that hTERT expression was 67.6%, 73.5%, and 100% in tumor, paratumor, and cirrhosis samples, respectively, but found no hTERT positivity in samples of normal liver. HBsAg, HBcAg, and HBxAg were expressed in 58.8%, 26.5%, and 76.5% of tumor tissues, respectively; in 64.7%, 41.2%, and 85.3% of the corresponding paratumor tissues; and in 76.7%, 66.7%, and 100% of cirrhotic tissues. The chi 2 test revealed no significant difference between the expression of hTERT and HBxAg in these tissues. Western-blot analysis revealed that expression of c-Myc and hTERT in the transiently transfected cells was much greater than that in the control cells. We elicited a similar result when we used the TRAP method to measure telomerase activity. Our data collectively demonstrate that HBX up-regulates the expression and activity of hTERT in hepatoma cells, suggesting that hTERT is associated with tumor development.

Upregulation of human telomerase reverse transcriptase mRNA expression byin vitrotransfection of hepatitis B virus X gene into human hepatocarcinoma and cholangiocarcinoma cells

To study the changes of human telomerase reverse transcriptase (hTERT) mRNA expression in human hepatocarcinoma cell lines (HepG2) and cholangiocarcinoma cell lines (QBC939) after HBx gene transfection and to illustrate the significance of transcriptional regulation of hTERT gene by HBx gene in the carcinogenesis. HepG2 and QBC939 cell lines were cultured and co-transfected with eukaryotic expression vector containing the HBx coding region and cloning vector containing enhanced green fluorescent protein (EGFP) coding sequence using lipid-mediated gene transduction technique. Thirty-six hours after transfection, EGFP expression in cells was used as the indicator of successful transfection. Flow cytometry was performed to determine the transfection efficiency. Cells were harvested and total RNA was extracted using TRIzol reagent. The expression of hTERT mRNA in HepG2 and QBC939 cell lines was assayed by reverse transcription-polymerase chain reaction. The expression of HBx protein in both cell lines was detected by immunocytochemical staining and Western blotting. Flow cytometry showed that the transfection efficiency was 46.4% in HepG2 cells and 29.6% in QBC939 cells for both HBx gene expression vector and blank vector. The expression of hTERT mRNA was meaningfully increased in HepG2 and QBC939 cell lines when transfected with HBx gene expression vector compared to those transfected with OPTI-MEM medium and blank vector. Immunocytochemical staining and Western blotting revealed HBx protein expression in HepG2 and QBC939 cells only when transfected with HBx gene. HBx gene transfection can upregulate the transcriptional expression of hTERT mRNA. The transactivation of hTERT gene by HBx gene is a newfound mechanism for pathogenesis of hepatocarcinomas and cholangiocarcinomas after HBV infection.

Detection and expression of hepatitis B virus X gene in one and two‐cell embryos from golden hamster oocytes in vitro fertilized with human spermatozoa carrying HBV DNA

The main objectives of this study were to introduce motile human sperm carrying Hepatitis B virus (HBV) DNA to golden hamster oocytes in a co-culture environment and to detect the replication and expression of the HBx gene in early embryonic cells. Zona-free hamster oocytes were inseminated with human sperm carrying pBR322-HBV DNA plasmid using the in vitro fertilization (IVF) technique. Both the one- and two-cell stages of early embryonic development were studied. PCR, RT-PCR, and Dot hybridization were performed to observe the HBx gene and its expression in these stages. "Fluorescence in situ hybridization" (FISH) was carried out to confirm the integration of HBV into the pronucleus, nucleus, and the chromosomes of embryos. The results showed that we have the ability to obtain a fertilization rate of 80%. RT-PCR showed that the HBx gene could be expressed in both one- and two-cell stages of embryonic development. The data suggested the possibility of sperm as a vector for the vertical transmission of HBV DNA to the next generation.

HBsAg and HBx knocked into the p21 locus causes hepatocellular carcinoma in mice

Hepatocellular carcinoma (HCC) affects males in a significantly higher proportion than females and is one of the human cancers etiologically related to viral factors. Many studies provide strong evidence of the direct role that hepatitis B virus (HBV) plays in hepatic carcinogenesis, but the functions of HBV surface antigen (HBsAg) and X protein (HBx) in hepatocarcinogenesis through direct or indirect mechanisms are still being debated. We generated two HBV gene knock-in transgenic mouse lines by homologous recombination. HBsAg and HBx genes were integrated into the mouse p21 locus. Both male and female p21-HBx transgenic mice developed HCC after the age of 18 months; however, male p21-HBsAg transgenic mice began to develop HCC 3 months earlier. The expression of a number of genes related to metabolism and genomic instability largely resembled the molecular changes during the development of HCC in humans. ER-beta (estrogen receptor-beta) was extremely up-regulated only in tumor tissues of male p21-HBsAg mice, providing genetic evidence that HBsAg might be the major risk factor affecting the gender difference in the causes of HCC. In conclusion, these mice might serve as good models for studying the different roles of HBsAg and HBx in early events of HBV-related hepatocarcinogenesis.

Involvement of extracellular signal-regulated kinase/mitogen-activated protein kinase pathway in multidrug resistance induced by HBx in hepatoma cell line

To investigate the molecular mechanism of the influence of HBx protein on multidrug resistance associated genes: multidrug resistance 1 (MDR-1), multidrug related protein (MRP-1), lung resistance related protein (LRP) in hepatoma cells and the potential role of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway in this process. A cell model stably expressing the HBx protein was established by liposome-mediated transfection of HBx gene into HepG2 cell line. The expression of multidrug resistance associated genes and proteins was detected by RT-PCR and Western blot. AnnexinV-FITC/PI assay was used to confirm the multidrug resistance (MDR) phenotype of transfected cells by fluorescence cytometry (FACS). The ERK/MAPK pathway activation was measured by Western blot through comparing the ratio of phosphorylation of ERK/MAPK to total ERK/MAPK protein. After treated with the ERK/MAPK pathway inhibitor U0126, the HBx-expressing cells were harvested. Then RT-PCR, Western blot and FACS were used to analyze the alterations in the expression of multidrug resistance associated genes and the MDR phenotype after exposure. Compared with the control group, the transfected cells showed a higher expression of MDR associated genes and proteins. Marked elevations in MDR-1 (64.3%), MRP-1 (87.5%) and LRP (90.8%) were observed in the transfected cells (P<0.05). RT-PCR revealed that the over-expression of MDR associated proteins was due to amplification of such genes (MDR1 2.9 fold, MRP1 1.67 fold, LRP1.95 fold). Furthermore, we found that the ERK/MAPK activity was remarkably high in the HBx-expressing cells. The activation of ERK/MAPK, as measured by the ratio of phosphorylated ERK bands normalized to the total ERK bands, was increased by 2.3-fold in HBx-transfected cells compared with cells transfected with the empty vector. After treated with the ERK/MAPK pathway inhibitor, the level of MDR associated genes and proteins in the transfected cells decreased to some extent. Compared with controls, a significant decrease in MDR-1 mRNA (53.3%), MRP-1 mRNA (59.7%) as well as LRP mRNA (56.4%) was observed in the U0126 treated transfected cells after 12 h. Western blot also demonstrated that the protein expression of these MDR associated genes slightly reduced after treated with U0126 for 12 h (MDR-1 40.1%, MRP-1 29.4%, LRP35.7%). This change was accompanied with the rise of cell apoptosis ratio confirmed by Annexin V-PI detection. The apoptosis index of U0126-treated cells increased by 1.28 fold, compared with that of transfected cells. Obviously, the MDR phenotype of these cells was obviously related with increased activities of the ERK/MAPK pathway. HBx protein might be one of the causes for the occurrence of MDR in HCC, and ERK/MAPK pathway might be involved in this change.

Interferon alpha inhibits the nuclear factor kappa B activation triggered by X gene product of hepatitis B virus in human hepatoma cells

X gene product of hepatitis B virus (HBV) (HBx) regulates many transcription factors including nuclear factor kappa B (NF-κB) and plays a key role in hepatocarcinogenesis. In this study, we demonstrated that the expression of full HBV genome and HBx gene similarly stimulated the transcriptional activity of NF-κB in HuH-7 human hepatoma cells, and that interferon (IFN)-α as well as dominant negative mutant of IκB kinase-α effectively inhibited the HBx-mediated NF-κB activation, but IFN-γ did not. These results suggest that IFN-α may have a function to block the NF-κB activating pathway triggered by HBx in HBV hepatocytes.