广西肝癌高发区原发性肝细胞癌 HBV X 基因的整合及影响因素分析

目的 了解广西肝癌高发区乙型肝炎病毒（hepatitis B virus,HBV）X区基因在肝细胞癌（hepatocellular carcinoma,HCC）染色体中的整合及影响因素。方法 以30例与HBV相关的原发性肝细胞癌患者为研究对象。提取HCC组织及癌旁组织标本DNA作为模板,以HBV X基因上游序列和人类基因组Alu重复序列为引物,应用重复序列-多聚酶链反应（Alu-PCR）扩增整合的HBV X片段及两侧的人类基因组DNA片段。扩增产物进行测序,计算目的片段整合率并分析相关的影响因素。结果 18例HCC组织检测到HBV X基因的整合片段,整合率为60.00%（18/30）;26例癌旁组织检测到HBV X基因的整合片段,整合率为86.67%（26/30）。癌旁组织HBV病毒整合率高于HCC组织,差异有统计学意义（χ²=5.445,P=0.020）。不同性别、年龄、HBeAg、HBV DNA、ALT、AST的HCC癌组织及其癌旁组织HBV X基因整合率比较差异均无统计学意义（P＞0.05）。结论 广西肝癌高发区癌旁组织比HCC组织HBV整合率高,说明HBV整合发生在感染早期。HBV X基因整合与HCC患者性别、年龄、HBeAg、HBV DNA、ALT、AST无明显关系。     

p16影响乙肝病毒相关性肝细胞肝癌的发生

目的　探讨乙肝病毒 (HBV)基因整合和p16基因表达改变及其与肝细胞肝癌发生发展的关系。方法　 35例肝癌及癌旁肝组织为标本 ,采用聚合酶链式反应 (PCR)及Southernblot检测HBVX基因整合 ,以单链构象多态性分析确定p16基因突变 ,以RT PCR检测p16mRNA ,以Westernblot检测p16蛋白。结果　肝癌中X基因整合与p16mRNA及蛋白表达相关 (P <0 .0 5 ) ,肝癌及癌旁肝组织中p16蛋白表达缺失率分别为 6 2 .9%和 4 0 .0 % ,差异有显著性 (P <0 .0 5 ) ;癌组织中p16蛋白表达缺失与肝癌分化程度、癌细胞浸润有相关性 (P <0 .0 5 )。结论　X基因整合与p16蛋白缺陷有关 ,p16改变在肝细胞癌变各阶段发挥重要作用 ,并与肝癌的演进和侵袭有关。     

Alteration of gene expression in human hepatocellular carcinoma with integrated hepatitis B virus DNA.

PURPOSE: Integration of hepatitis B virus (HBV) DNA into the human genome is one of the most important steps in HBV-related carcinogenesis. This study attempted to find the link between HBV DNA, the adjoining cellular sequence, and altered gene expression in hepatocellular carcinoma (HCC) with integrated HBV DNA. EXPERIMENTAL DESIGN: We examined 15 cases of HCC infected with HBV by cassette ligation-mediated PCR. The human DNA adjacent to the integrated HBV DNA was sequenced. Protein coding sequences were searched for in the human sequence. In five cases with HBV DNA integration, from which good quality RNA was extracted, gene expression was examined by cDNA microarray analysis. RESULTS: The human DNA sequence successive to integrated HBV DNA was determined in the 15 HCCs. Eight protein-coding regions were involved: ras-responsive element binding protein 1, calmodulin 1, mixed lineage leukemia 2 (MLL2), FLJ333655, LOC220272, LOC255345, LOC220220, and LOC168991. The MLL2 gene was expressed in three cases with HBV DNA integrated into exon 3 of MLL2 and in one case with HBV DNA integrated into intron 3 of MLL2. Gene expression analysis suggested that two HCCs with HBV integrated into MLL2 had similar patterns of gene expression compared with three HCCs with HBV integrated into other loci of human chromosomes. CONCLUSIONS: HBV DNA was integrated at random sites of human DNA, and the MLL2 gene was one of the targets for integration. Our results suggest that HBV DNA might modulate human genes near integration sites, followed by integration site-specific expression of such genes during hepatocarcinogenesis.     

HBV genome integration and genetic instability in HBsAg-negative and anti-HCV-positive hepatocellular carcinoma in Japan

The aim of this study is to clarify the existence and the form of HCV RNA and HBV DNA genome integration and genetic instability in liver tissue with HBsAg-negative and anti-HCV-positive HCC. We investigated 16 Japanese patients with HBsAg-negative and anti-HCV-positive HCC. HBV genome integration into host cell genome by Southern hybridization and PCR was examined. Moreover, we analyzed loss of heterozygosity (LOH) and replication errors (RER) of chromosomes 2p, 3p and 17p using the PCR and an autosequencer to determine the three microsatellite regions D2S123, D3S1067, TP53. Eight (50.0%) of 16 were found to have integrated genome of HBV in tumor tissue (T) by PCR. In even the non-tumor regions (NT), seven patients (43.8%) were found to have HBV genome integration. The coincidence between T and NT was found in 4 (25%). Integration of HBV-X gene in T was revealed in three (18.7%), and HBV-integration was confirmed in all NT. No integration of the X gene alone was found in the liver tissue. Five (37.5%) of eight HBV DNA integrated cases simultaneously had HCV RNA minus strand. Concerning the genetic instability, RER were detected in two of 16 (12.5%). RER at 2p; D2S123 was observed in one of 16 (6.2%) and at 3p; D3S1067 was observed in one (6.2%). LOH at the D2S123 locus was observed in one of 12 tumors with heterozygosity (8.3%). There was no genetic instability (LOH or RER) of TP53 which was p53 locus on 17p in T. There was only one case of eight HBV DNA integrated cases (6.2%) with genetic instability of RER of 3p simultaneously in T. In conclusion, the majority of HBsAg-negative and anti-HCV-positive HCC liver tissue was found to have HCV-RNA and HBV DNA integration, and in some samples, HBV DNA integration and genetic instability were concurrently confirmed. It is speculated that multistep carcinogenesis may have been proposed for HCC oncogenetic progression.     

Malignant transformation and EGFR activation of immortalized mouse liver epithelial cells caused by HBV enhancer-X from a human hepatocellular carcinoma

We have previously observed that all human hepatocellular carcinomas (HCCs) from HBV carriers examined had the integrated X region. In this study, HBV DNA was isolated from an integration site in one HCC that had a single, very small integrated viral DNA including the X region, but it had no expression of X gene as poly(A)RNA. It was found that HBV DNA was present between alphoid repetitive sequences, and it included Enhancer and X regions, encompassing the adr sequence from 910 to 1811. Nucleotides for 8 amino acids at the 3' end, a stop codon of X gene and a poly(A) signal downstream of X gene were lost by integration, and nucleotides for 7 amino acids and a stop codon were substituted by a connected alphoid sequence. When this cloned HBV DNA was transfected with an expression vector to an immortalized mouse liver epithelial cell line, MLE-10, malignant transformation occurred. Transformants having expressed poly(A)RNA of the X gene showed anchorage-independent growth in soft agar and tumor formation in the subcutis of nude mice. The mRNA level of EGFR was found to be remarkably enhanced in X-transformed cells, in contrast with the absence of this mRNA in parental and ras-transformed MLE-10. Our data provide evidence that the Enhancer-X region alone is the key contributor to the malignant change of pre-malignant liver cells in HBV carriers through activation of some specific genes, such as EGFR.     

原发性肝癌与乙型肝炎病毒X区基因变异的关系初探

目的 探讨乙型肝炎病毒X区基因变异与原发性肝癌的关系。方法 39例乙型肝炎病毒（HBV）感染患者的组织标本中,慢性肝病（CHB）14例、肝细胞癌（HCC）25例。采用聚合酶链反应（PCR）方法扩增组织中HBV X区基因,并对PCR产物进行DNA测序,分析常见变异位点的变异情况。结果 与CHB组相比,HCC组在X区发生插入或缺失变异及联合变异的位点及例数增多,并且A1762T与G1764A常发生双突变。HBV X 区变异频率较高位点依次为：C1655T／G、A1605C／G、A1762T、G1764A、A1772B、A1645C、C1687A、G1776T。结论 HBV X区存在点突变、插入或缺失变异及联合变异,可能在HCC的发生发展过程中起重要作用。     

Hepatitis B virus-induced hepatocellular carcinoma

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 signal pathways that associated with cell proliferation and apoptosis, and the impact of HBx C-terminal truncation in the development of HCC has been implicated. Recent studies by advanced sequencing technologies have revealed recurrent HBV DNA integration sites in hepatoma cells and susceptible genes/SNPs play an important role in the pathogenesis of liver cancer. Epigenetic changes, immune and inflammatory factors are also important contributing factors for liver cancer. This mini-review provides an overview on the recent development of HBV induced HCC.     

Clonal state of human hepatocellular carcinoma and non-tumorous hepatocytes

We determined the clonal state in specimens of hepatocellular carcinoma (HCC) and non-tumorous hepatocytes from the integration mode of hepatitis B virus (HBV) DNA. The integration mode of HBV DNA in several parts of the tumors and non-tumorous regions of the same liver, as well as in metastatic tumors, was examined using the Southern blot analysis. In 13 of the 14 cases of HCC, the liver tumors, including metastatic tumors in lymph nodes and the lungs, were monoclonal. In one case, a different HCC clone was found in one part of the liver tumor. The integration of HBV DNA was also observed in non-tumorous tissues in 38 of the 78 cases (49%) of chronic hepatitis with and without HCC; in 16 cases of chronic hepatitis in which HBV DNA was integrated, several clones of the hepatocytes that had HBV DNA integrated into their chromosomal DNA and had proliferated clonally were found in non-tumorous tissues. These clones were different from the tumor clone of the same liver. Thus HCCs were usually monoclonal. The development of different tumor clones appeared to be unusual, but the nontumorous hepatocytes could have proliferated clonally from different multicentric clones before carcinogenesis. The clonal growth of the non-tumorous hepatocytes suggests that the integration of HBV DNA plays an important role in hepatocarcinogenesis.     

USING 3 PRIMER PAIRS TO DETECT HBV DNA IN LIVERTISSUES FROM HEPATOCELLULAR CARCINOMASWITH PCR TECHNIQUE

ＵＳＩＮＧ３ＰＲＩＭＥＲＰＡＩＲＳＴＯＤＥＴＥＣＴＨＢＶＤＮＡＩＮＬＩＶＥＲＴＩＳＳＵＥＳＦＲＯＭＨＥＰＡＴＯＣＥＬＬＵＬＡＲＣＡＲＣＩＮＯＭＡＳＷＩＴＨＰＣＲ　ＴＥＣＨＮＩＱＵＥＣｈｅｎＭｉｎｇ陈明；ＬｕＬｉｎｇ吕凌；ＹａｏＪｉｌｕ姚集鲁；Ｐｅｎｇ...     

原发性肝细胞癌组织中乙肝病毒整合与p53基因突变关系研究

应用Ｓｏｕｔｈｅｒｎ杂交方法检测１５例原发性肝细胞癌及癌旁组织乙肝病毒（ＨＢＶ）存在状态，采用ＰＣＲ－ＳＳＣＰ（Ｐｏｌｙ－ｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎ－ｓｉｎｇｌｅｓｔｒａｎｄｃｏｎｆｏｒｍａｔｉｏｎｐｏｌｙｍｏｒｐｈｉｓｍ）银染技术研究其ｐ５３基因突变。结果癌组织中１３例（８６．７％）有ＨＢＶＤＮＡ整合，同时３例伴游离ＨＢＶＤＮＡ；癌旁４例（２６．７％）有ＨＢＶＤＮＡ整合，而游离的ＨＢＶＤＮＡ却有６例。癌组织中ｐ５３基因变异８例（５３．３％），１例位于第５外显子，各有２例位于第６、第７外显子，３例位于第８外显子，癌旁组织未见ｐ５３基因异常，ｐ５３基因异常的癌组织中均有ＨＢＶＤＮＡ整合。研究结果表明，ＨＢＶＤＮＡ整合与ｐ５３基因突变密切相关，ＨＢＶＤＮＡ可能通过整合基因组后导致抑癌基因突变失去抑癌作用而引发肝癌。     

Identification and characterization of a prevalent hepatitis B virus X protein mutant in Taiwanese patients with hepatocellular carcinoma

The aim of this study was to investigate whether there was a particular hepatitis B virus (HBV) X protein (HBx) mutant associated with Taiwanese patients with hepatocellular carcinoma (HCC). Initially, the entire coding region of HBx gene from the serum samples of 14 Taiwanese patients were sequenced. A novel mutant, HBx-A31, was preferentially found in patients with HCC. Sera from 67 patients with HCC and 100 patients with chronic hepatitis B were thus subjected for codon 31 analysis using a dual amplification created restriction site method. HBx-A31 was detected more frequently in patients with HCC (52% versus 12%; P<0.001) and in patients with liver cirrhosis (44% versus 6%; P<0.001). Site directed mutagenesis experiment revealed that HBx-A31 was less effective in transactivating HBV enhancer I-X promoter complex, less efficient in supporting HBV replication, and less potent in enhancing TNF-alpha induced increment of CPP32/caspase 3 activities in HepG2 cells. In conclusion, a prevalent HBx mutant was identified in Taiwanese patients with hepatocellular carcinoma. Development of this mutant might represent a strategy of the virus to escape immune surveillance and thus contribute to the process of multiple-step hepatocarcinogenesis.     

Surface,core, and X genes of hepatitis B virus in hepatocellular carcinoma: an in situ hybridization study

BACKGROUND: The incidence of hepatocellular carcinoma (HCC) and the seroprevalence of hepatitis B virus (HBV) in this disease state are significantly higher in South India than in North India. Because data on serologic studies do not project the actual association between the two parameters, this study was undertaken. METHODS: The prevalence of HBV genes in HCC patients was studied using nonisotopic in situ hybridization. Fifty patients from South India were diagnosed with HCC after performing ultrasound-guided fine-needle aspiration biopsies of liver lesions. The diagnosis was confirmed by cell block studies. Sections cut from paraffin-embedded cell blocks made out of the aspirates were probed with digoxigenin-labeled surface, core, and X regions of the viral genome. RESULTS: Nuclear integration of the surface gene was observed in 100% (50 of 50), the core gene was positive in 94% (47 of 50), and the X gene was present in 98% (49 of 50) of the cases. An episomal form of the virus was not found. Serum hepatitis B surface antigen was positive only in 48% (12 of 25) of the patients screened. CONCLUSIONS: We found molecular evidence that HBV is an important contributing factor in the etiology of HCC in South India. In HCC, the S gene of the virus was the most prevalent followed by the X and C genes. Only integrated forms of the viral DNA were observed. Nonisotopic in situ hybridization using multiple regions of the viral genome is a good technique for studying this association. It has an added advantage over polymerase chain reaction, of localization of signals in a tumor cell. Cell blocks made from fine-needle aspirates are ideal for in situ hybridization.     

Hepatitis B virus DNA integration frequently observed in the hepatocellular carcinoma DNA of hepatitis C virus-infected patients

Human hepatitis B virus (HBV) and hepatitis C virus (HCV) are two major etiologic agents of chronic hepatitis, which is closely related to the development of hepatocellular carcinoma (HCC). We investigated the role of HBV coinfection in ongoing HCV-related liver diseases in HCV-infected patients. We found a high prevalence of anti-HBc in anti-HCV-positive/HBsAg-negative HCC patients and also found a close correlation between anti-HBc positivity and integration of HBV DNA into HCC DNA of anti-HCV-positive/HBsAg-negative patients. The present data suggest that integrated HBV DNA may play an important role in the development of HCC in the anti-HCV-positive/HBsAg-negative patients carrying the anti-HBc antibody.     

Integration of the hepatitis B virus X fragment in hepatocellular carcinoma and its effects on the expression of multiple molecules: a key to the cell cycle and apoptosis

We investigated hepatitis B virus (HBV) DNA integration and expression of several proteins involved in the cell cycle and apoptosis, including cyclin A, retinoblastoma protein (pRB), Fas-associated death domain protein (FADD), tumor necrosis factor receptor-associated death domain protein (TRADD), and nuclear factor kappaB (NF-kappaB) in HBV-associated hepatocellular carcinoma (HCC) and liver cirrhosis (LC). Archival HCC and LC specimens were obtained from 35 patients each with HBV infection; 5 normal liver specimens used as controls were also obtained. Polymerase chain reaction and Southern blot hybridization were used to detect the integration of HBV DNA in the HCC and LC specimens. The protein levels were determined by Western blot assay. The difference in HBV DNA integration between HCC and LC and correlation between HBV-encoded X protein (Hbx) integration and protein expression were analyzed statistically. HBV DNA was detected in 33 (94%) of the HCC and LC specimens. HBx integration differed in the HCC [24 (69%)] and LC [14 (40%)] specimens (p=0.015). Sixty percent of the HCC specimens and 6% of the LC specimens had increased cyclin A expression. Also, 34, 37, 69, and 77% of the HCC specimens were positive for pRB, FADD, TRADD, and NF-kappaB expression, whereas 80, 60, 100, and 100% of the LC specimens were positive for pRB, FADD, TRADD, and NF-kappaB expression. Significant correlations between HBx integration and the level of expression of cyclin A (r=0.452; p=0.006), pRB (r=-0.419; p=0.012), and TRADD (r=0.470; p=0.004) were discovered. Therefore, integration of HBV DNA occurred frequently in HCC and LC cases with chronic HBV infection, whereas HBx integration occurred more often in HCC than in LC cases (p=0.015). HBx integration and altered expression of genes is a key to apoptosis and may play important roles in HBV-induced hepatocarcinogenesis.     

The role of previous infection of hepatitis B virus in Hbs antigen negative and anti-HCV negative Japanese patients with hepatocellular carcinoma: etiological and molecular biological study

The aim of this study is to elucidate the important role of the previous infection of HBV, and the relations among HBV genome integration and p53 gene mutation, telomerase activity and genetic instability in liver tissue with HBsAg-negative (NB) and anti-HCV negative (NC) hepatocellular carcinoma (HCC). We examined the backgrounds of 34 NB and NC (NBNC) Japanese patients with chronic liver disease (CLD) patients not associated with HCC and 26 NBNC CLD patients with HCC. HBV genome integration into host cell genome, p53 gene mutation telomerase activity and genetic instability were examined in 6 with NBNC HCC (NBNC-HCC) tumorous tissue (T) and non-tumorous tissues (NT). In the NBNC group, HBV-related antibody positive patients with HCC are significantly more than the patients without HCC. Moreover, concerning the stage of the coexisted liver diseases, in NBNC CLD, LC patients with HCC is 19 of 26 (73.1%) , on the other hand, LC patients without HCC is 16 of 34 (47.1%). LC patients with HCC group is significantly more than that without HCC. Three (50%) of 6 in T and 3 cases (50% ) in NT were found to integrated genome of HBV. p53 gene mutation was observed in 3 (50%) of T. Concerning the telomerase activity, 3 of 6 cases (50%) in T and 1 case in NT was recognized. There was no genetic instability (LOH or RER) of D2S123, D3S1067 and TP 53 in T and NT. Finally in T of NBNC HCC cases, TTVDNA was detected in 3 of 5. Even in the HBsAg-negative and anti-HCV negative HCC cases, CLD coexisting with LC, previous HBV infection and HBVDNA integration were observed. There were a few cases with HBVDNA integration, p53 gene mutation, telomerase activity and genetic instability, simultaneously in HCC tissue, and in some cases, the coexistence with TTVDNA were concurrently confirmed. It is speculated that the important role of the previous infection of HBV may have also been proposed for HCC oncogentic progression in NBNC CLD [corrected].     

Liver diseases associated with hepadnavirus infection. A study in duck model

Among four hepadnaviruses, we studied liver diseases of duck hepatitis B virus (HBV)-infected animals. There was close correlation of viral infection and development of liver diseases. The presence of hepatocellular carcinoma (HCC) was observed in Chinese ducks. We previously showed that duck HBV DNA was integrated in the neoplastic cells. Analysis of duck HBV integrants revealed the similarity to the reported structures of HBV integrants. To develop effective antiviral therapy, we tested adenine arabinoside (Ara-A), phosphonoformate, azido-deoxythymidine (AZT), and dideoxycytidine (DDC) in this in vivo model. Significant suppressive effects on viral replication were observed by phosphonoformate and adenine arabinoside, but AZT and DDC failed to show significant antiviral effects. This may be related to a unique pathway of replication in hepadnaviruses.     

肝细胞癌患者B和C基因型乙型肝炎病毒X蛋白的变异特点

背景与目的：X蛋白是乙型肝炎病毒(hepatitis B virus,HBV)最重要的致病因子之一,它在HBV相关性肝细胞癌(hepatocellular carcinoma,HCC)的发生发展中起着重要作用。目前已知B、C基因型HBV相关性HCC的临床及病理表现不尽相同,但目前尚不清楚这种差别是否与B、C基因型HBV X基因之间的差别有关。因此本实验拟研究B、C基因型间HBV X蛋白氨基酸差异及其在HCC患者中的变异及特点,初步探讨其与HCC发生、发展的关系。方法：PCR扩增22份乙型肝炎病毒表面抗原(HBsAg)阳性HCC患者血清HBV X基因,克隆、测序并以Vector NTI6．0软件分析其基因型。以DNAMAN软件对标准HBV及HCC来源的HBV X蛋白进行氨基酸同源分析。结果：检测的22个HBV X基因片段均属于B或C基因型。B、C基因型HBV X蛋白之间存在14个氨基酸的差异,HCC患者来源的B、C型HBV X蛋白存在4个氨基酸的共有变异,C型HBV X蛋白尚有6个型特异性变异。这些差异或变异氨基酸均位于X蛋白B、T细胞表位或反式激活区及调节区内。结论：B、C基因型HBV X蛋白之间存在氨基酸差异,且在HCC中发生基因型特异性变异,这些差异或变异氨基酸可能导致X蛋白免疫学功能及反式激活功能的不同。     

Hepatitis B virus-related insertional mutagenesis in chronic hepatitis B patients as an early drastic genetic change leading to hepatocarcinogenesis

Growing evidence demonstrates that hepatitis B virus (HBV) integration and resulting insertional mutagenesis play an important role in cell growth or maintenance in hepatocellular carcinomas (HCCs). To determine if HBV integration occurs and affects cellular genes at such a stage of infection, we analysed viral-host junctions in chronic hepatitis tissues without HCC using PCR amplification with primers specific to human Alu-repeat and HBV. We obtained 42 independent viral-host junctions from six patients examined and identified chromosomal locations for 20 of the 42 junctions. In six clones, each integration apparently affected a single gene. These six candidate genes included one known tumor suppressor gene, three human homologs of drosophila genes that are critical for organ development, one putative oncogene and one recently found chemokine. Our data, together with previously reported HBV integrants in HCCs, suggested preferential HBV integration into chromosome 3 (P = 0.022). Our virus-tagging approach provided (a) firm evidence of HBV integration in hepatocytes at an early stage of chronic infection and (b) revealed cellular genes possibly affected by HBV integration and potentially involved in early steps of the process leading to carcinogenesis.