乙型肝炎病毒X蛋白在原发性肝癌中的作用

乙型肝炎病毒X蛋白(HBx)是一种由HBV X基因编码的多功能蛋白,参与调节基因转录、细胞信号转导、细胞增殖转化、细胞周期和细胞凋亡。目前HBx蛋白被认为在HBV诱发原发性肝癌的过程中发挥重要作用。介绍了HBx蛋白与癌基因、抑癌基因、细胞增殖、细胞凋亡、肝癌侵袭转移和肝癌干细胞的关系,探讨了HBx蛋白在肝癌发生发展中的作用。     

Hepatitis B virus X protein: a multifunctional viral regulator

Received: May 31, 2001 / Accepted: June 29, 2001     

Differential integration rates of hepatitis B virus DNA in the liver of children with chronic hepatitis B virus infection and hepatocellular carcinoma

BACKGROUND AND AIM: Integration of hepatitis B virus-DNA (HBV-DNA) into the host genome, a phenomenon found frequently in hepatocellular carcinomas (HCC) and causally linked to oncogenesis, has not been well characterized in children. The aim of the present study was to determine the prevalence of HBV integration more accurately and to decide whether the integration rate varies at different stages of chronic HBV infection in children. METHODS: Of 13 children with chronic hepatitis, 14 liver biopsy tissues were analyzed. One liver tissue with pure liver cirrhosis, nine non-tumor, and nine tumor liver tissues from children with HCC were analyzed by a very sensitive method, inverse polymerase chain reaction (IPCR). RESULTS: Thirteen genuine viral-host junctional sequences from 23 patients were successfully isolated and proved that IPCR is a useful method in this context. The results also indicated that the detection rate of HBV-DNA integration increased in parallel with the progress of liver histology towards the neoplastic transformation, with 0% in the liver of chronic hepatitis, 22.2% in non-tumor livers of HCC patients, and 66.7% in tumor liver tissues of HCC patients. CONCLUSION: The present results indicate that integration of HBV-DNA into the host genome was rarely confirmed at the early stage of chronic hepatitis in children until the stage of HCC formation.     

Hepatitis B virus markers and antibodies to hepatitis C virus in Japanese patients with hepatocellular carcinoma

Sera from Japanese patients with chronic liver disease were tested for hepatitis B virus (HBV) markers and antibodies to hepatitis C virus (anti-HCV), and the results were correlated to the presence of hepatocellular carcinoma. In chronic non-A, non-B liver disease, anti-HCV prevalence was high both in patients with hepatocellular carcinoma (78/89, 88%) and without it (66/84, 79%), while previous HBV infection was more common in patients with hepatocellular carcinoma (65/89, 73%) than in those without it (46/84, 55%) (P<0.05). Coexistence of anti-HCV and antibodies to HBV was observed frequently in patients with hepatocellular carcinoma (56/89, 63%) compared with patients without it (39/84, 46%) (P<0.05). In chronic HBV carriers, anti-HCV was more common in patients with hepatocellular carcinoma (12/38, 32%) than in those without it (3/62, 5%) (P<0.01). These results suggest that infection with the two viruses may be a risk factor for more serious liver disease.This work was supported by a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan.     

稳定表达乙型肝炎病毒X基因肝细胞株的构建

目的 构建稳定表达乙型肝炎病毒x基因（HBVx）的正常人肝细胞株。方法用PCR法扩增含EcoRI和HindⅢ酶切位点的x基因序列,构建HBVx基因真核表达载体pcDNA3．1（＋）-HBVx,转化大肠杆菌DHSct,筛选阳性克隆并对其进行双酶切和测序鉴定;用脂质体转染法将HBVx基因导入Chang细胞系,G418筛选,RT-PCR和Westernblot鉴定。结果x基因亚克隆入pcDNA3．1（＋）,含有完整的x基因片段,转染后Chang细胞有HBVxmRNA表达,Chang／HBVx有HBVx蛋白的表达。结论构建了稳定表达HBVx基因的肝细胞株Chang／HBVx。     

Basal core-promoter mutant of hepatitis B virus and progression of liver disease in hepatitis B e antigen-negative chronic hepatitis B.

BACKGROUND/AIMS: The long-term outcomes in hepatitis B e antigen (HBeAg)-negative chronic hepatitis B are distinct from those in HBeAg-positive chronic hepatitis. However, the molecular virological factors that contribute to the progression of liver disease in this special clinical setting remain largely unknown. We thus investigated the association of hepatitis B virus (HBV) genotypes as well as precore/basal core-promoter mutations with the clinical and virological characteristics of patients with HBeAg-negative chronic hepatitis B in Taiwan. METHODS: HBV genotypes and sequences of precore and basal core-promoter regions of the HBV genome were determined in 174 HBeAg-negative chronic HBV infection patients including 62 inactive carriers and 112 with different stages of liver disease. RESULTS: HBV carriers with older age (> 50 years) (odds ratio, 9.09; 95% confidence interval (CI), 3.22-25, P < 0.001) and basal core-promoter mutant of HBV (odds ratio, 4.12; 95% CI, 1.41-12.03, P = 0.01) were associated with the development of liver cirrhosis and hepatocellular carcinoma (HCC). The gender-related risk factors associated with the development of liver cirrhosis and HCC were further analyzed, and basal core-promoter mutant was only associated with the development of liver cirrhosis and HCC in male carriers (odds ratio, 4.35; 95% CI, 1.30-14.52, P = 0.02). CONCLUSIONS: The risk of development of liver cirrhosis and HCC is significantly increased in patients with advanced age as well as with basal core-promoter mutant of HBV. In addition, basal core-promoter mutant might contribute to the gender difference of the progression of liver disease in HBeAg-negative chronic hepatitis B in Taiwan.     

Molecular mechanism of hepatitis B virus X protein function in hepatocarcinogenesis

Many factors are considered to contribute to hepatitis B virus(HBV)-associated hepatocellular carcinoma(HCC),including products of HBV,HBV integration and mutation,and host susceptibility. HBV X protein(HBx) can interfere with several signaling pathways associated with cell proliferation and invasion,and HBx C-terminal truncation has been suggested to impact the development of HCC. This review focuses on the pathological functions of HBx in HBV-induced hepatocarcinogenesis. As a transactivator,HBx can affect regulatory non-coding RNAs(nc RNAs),including micro RNAs and long nc RNAs. HBx is also involved in epigenetic modification and DNA repair. HBx interacts with various signal-transduction pathways,such as the p53,Wnt,and nuclear factor-κB pathways. We conclude that HBx hastens the development of hepatoma.     

Impact of occult hepatitis B virus infection and prior hepatitis B virus infection on development of hepatocellular carcinoma in patients with liver cirrhosis due to hepatitis C virus

Objective. Although hepatitis B virus (HBV) DNA can be detected in liver or sera of patients without serum hepatitis B surface antigen (HBsAg), its clinical relevance in hepatocarcinogenesis remains controversial. This observational cohort study was conducted to clarify the risk factors, including the presence of serum HBV DNA and hepatitis B core antibody (anti-HBc), for hepatocellular carcinoma (HCC) in patients with hepatitis C virus (HCV)-related liver cirrhosis (LC). Material and methods. The study comprised 123 patients with LC due to HCV, and negative for HBsAg. The risk factors for HCC development were analyzed by univariate and multivariate analysis. Serum samples were assayed for HBV DNA using real-time polymerase chain reaction. Results. Serum HBV DNA was detectable in 14 patients (11.4%) and serum anti-HBc in 96 (78.0%). During the follow-up period (mean 53.3 months), 80 patients (65.0%) developed HCC. The cumulative HCC development rate was significantly higher in the anti-HBc-positive group than in the anti-HBc-negative group (p=0.0039), but did not differ between the serum HBV DNA-positive and -negative groups (p=0.8570). The multivariate analysis indicated that male gender, alpha-fetoprotein (AFP) 20 ng/ml or greater, average serum alanine aminotransferase (ALAT) 80 IU/l or greater and the presence of anti-HBc were independent risk factors for development of HCC (p=0.038, p=0.013, p=0.020 and p=0.001, respectively). Conclusions. Serum anti-HBc, which indicates a previous HBV infection, has clinical significance in hepatocarcinogenesis in patients with HCV-related LC, but serum HBV DNA does not. Therefore, anti-HBc in serum is a significant predictor for HCC.     

Diabetes mellitus in chronic hepatitis B and C: prevalence and potential association with the extent of liver fibrosis

Diabetes mellitus has been reported to have an increased prevalence and to be associated with more severe fibrosis in patients with chronic hepatitis C. We evaluated the prevalence of diabetes mellitus in patients with chronic hepatitis B or C as well as the possible association between presence of diabetes and extent of liver fibrosis. In total, 434 consecutive patients with histologically documented hepatitis B e antigen (HBeAg)-negative chronic hepatitis B (n = 174) or chronic hepatitis C (n = 260) were studied. The relationships of diabetes and epidemiological, somatomorphic, laboratory and histological patient characteristics were evaluated. Liver histological lesions were blindly evaluated according to the Ishak's classification. Diabetes was present in 58 (13%) patients, without any difference between those with chronic hepatitis B (14%) or C (13%). Diabetes was observed significantly less frequently in patients with fibrosis score 0-2 (7.7%) than 3-4 (10.4%) than 5-6 (29.2%) (P < 0.001). The presence of diabetes was independently associated with higher gamma-glutamyl-transpeptidase (GGT) levels and more severe fibrosis or presence of cirrhosis (P < 0.001) as well as with presence of hepatic steatosis and increased serum triglycerides levels (P < 0.02). In the noncirrhotic patients, diabetes was significantly associated with older age and higher GGT levels, but not with the extent of fibrosis. In conclusion, diabetes mellitus is observed in more than 10% of patients with either HBeAg-negative chronic hepatitis B or chronic hepatitis C. The presence of diabetes is strongly associated with more severe liver fibrosis, but such an association may be related to the high prevalence of diabetes in patients with cirrhosis.     

Expression of nuclear factor-kappa B in hepatocellular carcinoma and its relation with the X protein of hepatitis B virus

AIM: In this study we investigated the relationship of the X protein of HBV and nuclear factor-kappa B (NF-kappa B) and the expression of NF-kappa B in human hepatocellular carcinoma tissues. METHODS: Immunohistochemistry SP method was used to detect the expression of NF-kappa B and the X protein of HBV in human hepatocellular carcinoma tissues of 52 cases. Gene transfection mediated by lipofectamine was used to transfect the eukaryotic expression vector pCDNA3.1-HBX of HBV x gene into human hepatocellular carcinoma cell line HCC-9204 and NF-kappa B was detected. RESULTS: NF kappa B was widely expressed in human hepatocellular carcinoma tissues in a total of 52 cases and its expression was related to the X protein of HBV. NF-kappa B was localized both in the cytoplasm and the nuclei of hepatocellular carcinoma cells in 11 cases which were positive for the X protein of HBV while in 41 cases negative for the X protein of HBV, NF-kappa B was only localized in the cytoplasm of hepatocellular carcinoma cells but translocated to the nuclei of hepatocellular carcinoma cells after the eukaryotic expression vector pCDNA3.1-HBX was transfected into HCC-9204 cells. CONCLUSION: This study strongly suggests that the nuclear factor NF-kappa B is widely expressed in hepatocellular carcinoma tissues in different styles according to the expression of the X protein of HBV. NF-kappa B is abnormally activated in hepatocellular carcinoma, which is probably related to the X protein of HBV. The X protein of HBV can activate NF-kappa B to translocate into nuclei of hepatocellular carcinoma cells.     

Expression of B7-H4 and hepatitis B virus X in hepatitis B virus-related hepatocellular carcinoma

AIM: To investigate the expression and clinical significance of B7-H4 and hepatitis B virus X(HBx) protein in hepatitis B virus-related hepatocellular carcinoma(HBV-HCC).METHODS: The expression of B7-H4 in the human HCC cell lines Hep G2 and Hep G2.2.15 were detected by western blot, flow cytometry, and immunofluorescence. The expression of B7-H4 and HBx in 83 HBV-HCC was detected by immunohistochemistry, and the relationship with clinicopathological features was analyzed. Paraffin sections were generated from 83 HBV-HCC patients(22 females and 61 males) enrolled in this study. The age of these patients ranged from 35 to 77 years, with an average of 52.5 ± 11.3 years. All experiments were approved by the Ethics Committees of the Second Affiliated Hospital, Zhejiang University School of Medicine.RESULTS: B7-H4 was significantly upregulated in Hep G2.2.15 cells compared to Hep G2 cells. Specifically, the protein expression of B7-H4 in the lysates of Hep G2 cells was more than that in Hep G2.2.15 cells. In addition, HBx was expressed only in Hep G2.2.15 cells. Similar data were obtained by flow cytometry. The positive rates of B7-H4 and HBx in the tissues of 83 HBV-HCC patients were 68.67%(57/83) and 59.04%(49/83), respectively. The expression of HBx was correlated with tumor node metastases(TNM) stage, and the expression of B7-H4 was positively correlated with HBx(rs = 0.388; p 0.01). The expression level of B7-H4 in HBx-positive HBV-HCC tissues was substantially higher than that in HBx-negative HBV-HCC tissues. The expression level of B7H4 was negatively related to tumor TNM stage.CONCLUSION: Higher expression of HBx and B7-H4 was correlated with tumor progression of HBV-HCC, suggesting that B7-H4 may be involved in facilitating HBV-related hepatocarcinogenesis.     

Occult hepatitis B virus infection and its clinical implications

Occult hepatitis B virus (HBV) infection is characterized by presence of HBV infection with undetectable hepatitis B surface antigen (HBsAg). Serum HBV level is usually less than 10⁴ copies/mL in these patients. Diagnosis of occult HBV infection requires sensitive HBV-DNA PCR assay. Several possibilities have been hypothesized as the mechanisms of occult HBV infection. These include: (i) mutations of HBV-DNA sequence; (ii) integration of HBV-DNA into host's chromosomes; (iii) infection of peripheral blood mononuclear cells by HBV; (iv) formation of HBV-containing immune complex; (v) altered host immune response; and (vi) interference of HBV by other viruses. The precise prevalence of occult HBV infection remains to be defined. The clinical implications of occult HBV infection involve different clinical aspects. First of all, occult HBV infection harbours potential risk of HBV transmission through blood transfusion, haemodialysis, and organ transplantation. Second, it may serve as the cause of cryptogenic liver disease, contribute to acute exacerbation of chronic hepatitis B, or even fulminant hepatitis. Third, it is associated with development of hepatocellular carcinoma. Fourth, it may affect disease progression and treatment response of chronic hepatitis C. Most of the previous studies utilized retrospective observation without control groups, and lacked direct association of occult HBV infection with specific pathological changes and disease progression. Highly sensitive, quantitative, and functional molecular analyses of HBV, combined with a well-designed prospective clinical assessment will provide the best approach for the future study of occult HBV infection.     

Aberrant epigenetic modifications in hepatocarcinogenesis induced by hepatitis B virus X protein

BACKGROUND & AIMS: The involvement of the hepatitis B virus X (HBx) protein in epigenetic modifications during hepatocarcinogenesis has not been previously characterized. The aim of the present study was to identify the involvement of HBx in regional hypermethylation and global hypomethylation during the formation of hepatocellular carcinoma (HCC). METHODS: Liver cell lines were transiently or stably transfected with an HBx-expressing vector. DNA methyltransferase (DNMT) promoter activity changes were examined by luciferase assay and chromatin immunoprecipitation. The methylation status of insulin-like growth factor binding protein-3 was examined by methyl-specific polymerase chain reaction and bisulfite sequencing. Global DNA methylation levels were examined using 5-methylcytosine dot blot and methylation-sensitive Southern blot analysis. HBx-mediated DNA methylation abnormalities were confirmed in patient HCC samples using methyl-specific polymerase chain reaction and 5-methylcytosine dot blot analysis. RESULTS: HBx expression increased total DNMT activities by up-regulation of DNMT1, DNMT3A1, and DNMT3A2 and selectively promoted regional hypermethylation of specific tumor suppressor genes. HBx specifically repressed insulin-like growth factor-3 expression through de novo methylation via DNMT3A1 and DNMT3A2 and by inhibiting SP1 binding via recruiting methyl CpG binding protein 2 to the newly methylated SP1 binding element. HBx also induced global hypomethylation of satellite 2 repeat sequences by down-regulating DNMT3B. The prevalence of these specific methylation abnormalities by HBx was significantly correlated with HBx expression in HBV-infected HCC patients. CONCLUSIONS: Targeted deregulation of DNMTs by HBx promotes both specific regional hypermethylation and global hypomethylation. These epigenetic modulations by HBx may suggest a mechanism for epigenetic tumorigenesis during HBV-mediated hepatocarcinogenesis.     

Clinical management of hepatitis B virus infection correlated with liver transplantation

BACKGROUND:As a radical cure for post-hepatitis B virus (HBV)-related liver cirrhosis and hepatocellular carcinoma,liver transplantation has been applied in many medical centers.Before the use of effective measures,hepatitis B recurrence and the existence of HBsAg(+) donors,patients with hepatitis B-related diseases are contraindicated for liver transplantation.Application of interferon,hepatitis B immunoglobulin (HBIG),and nucleotide analogues (e.g.,lamivudine) has made great progress in the clinical care ...     

HBsAg-negative hepatitis B virus infections in hepatitis C virus-associated hepatocellular carcinoma

This study was conducted to evaluate reports that hepatitis B virus (HBV) DNA sequences can be found in the serum and/or tumour tissue from some hepatocellular carcinoma (HCC) patients who have no detectable hepatitis B surface antigen (HBsAg) in their sera. Such HBV infections would be highly atypical, because prospective studies have shown a clear succession of specific serologic markers during and after most HBV infections. As most HBsAg-negative HCC patients in Japan have hepatitis C virus (HCV) infections, the present study was conducted to determine whether some of these patients actually have unrecognized HBV infections. Thirty newly diagnosed HCC patients from Kurume, Japan, with antibody to the hepatitis C virus (anti-HCV) were studied. None of the 30 had HBsAg detectable in their serum. Of 22 for whom test results for antibodies to the hepatitis B core antigen (anti-HBc) and antibodies to HBsAg (anti-HBs) were available, 14 (64%) had anti-HBc and anti-HBs, four (18%) had anti-HBc alone, and four (18%) had no HBV markers. Nested polymerase chain reaction was used to detect the HBV surface (S), core (C), polymerase (P) and core promoter gene sequences in the HCC tissues and in the adjacent nontumorous liver tissues. HBV DNA was detected in HCC and/or adjacent nontumorous liver in 22 of 30 (73%) patients [detected in both HCC and nontumorous liver in 19/30 patients (63%)]. Among the 22 patients with detectable HBV DNA, more than one HBV gene was detected in 10 (46%). Among the four patients whose sera were negative for all HBV markers, three had HBV DNA in either HCC and nontumorous liver (two cases) or only in the nontumorous liver (one case); HBV DNA could not be detected in tissues from the fourth patient. In 18 of 21 (86%) patients with detectable HBV core promoter sequences, mutations at both nucleotides 1762 (A-GT) and 1764 (G-A) in the core promoter region were found. No deletions were detected in the core promoter gene region of the type reported to be associated with some cases of HBsAg-negative HBV infection. Thus, HBV DNA was detectable in 22 (73%) HBsAg-negative, anti-HCV-positive HCCs, including three (10%) who were also negative for anti-HBc and anti-HBs. HBV mutations at both nucleotides 1762 (A-GT) and 1764 (G-A) in the core promoter region were found in the majority of cases, mutations that have previously been reported in HBV that is integrated in HCC DNA. In serologic surveys to determine etiologic associations of HCC, patients such as those in this study would have been incorrectly designated as having 'HCV-associated HCC,' whereas the data in this study suggest that HBV could have played a role in the development of their HCCs.     

Low frequency of mutations in the X gene, core promoter and precore region of hepatitis B virus infected Vietnamese

Numerous mutations in the hepatitis B virus (HBV) genome have been described, but in most cases their role in the pathogenesis of HBV infection is still unclear. Therefore, we analysed specific mutations in HBV-infected Vietnamese patients and assessed their potential relationship with their clinical outcome. A total of 153 HBV-infected Vietnamese patients with well-characterised clinical profiles were enrolled. None of the study participants had a history of alcohol or drug use and none received any antiviral or immunosuppressive therapy before or during the course of this study. The HBx- and core promoter regions were analysed by sequencing. The majority of isolates corresponded to genotype A. The presence of hepatitis B e antigen (HBeAg) was associated with significantly higher viral loads in the chronic HBV-infection group (P = 0.026). Double mutations in the core promoter (1762/1764) were more frequent in those with cancer than in noncancer patients (P < 0.01). Mutations at nucleotide (nt) 1766/1773 were found at low prevalence but with no obvious association to clinical presentation. Cytosine at nt 1858 was predominant but the stop codon mutation in the precore region was not detected. In the study, 4/48 hepatocellular carcinoma (HCC) patients revealed truncated HBx, whilst the serine to alanine mutation (codon 31) of HBx was more prevalent in cancer patients than in asymptomatic HBV carriers (P < 0.01). Thus, the low frequency of mutations indicates the relation of the absence of antiviral pressure in this population. The exclusively found prevalence of certain mutations detected in those with HBV-related carcinoma nevertheless indicates a degree of association with disease progression.