Evaluation of hepatitis B viral replication and proteomic analysis of HepG2.2.15 cell line after knockdown of HBx

BACKGROUND:Hepatitis B virus (HBV) is one of the major pathogens of human liver disease.Studies have shown that HBV X protein (HBx) plays an important role in promoting viral gene expression and replication.In this study we performed a global proteomic profiling to identify the downstream functional proteins of HBx,thereby detecting the mechanisms of action of HBx on virion replication.METHODS:HBx in the HepG2.2.15 cell line was knocked down by the transfection of small interfering RNA (siRNA).The replicati...     

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.     

乙型肝炎病毒X蛋白在肝细胞癌发生发展中的作用

乙型肝炎病毒(HBV)感染是急慢性病毒性肝炎的主要致病因素,它也是诱发肝硬化,原发性肝细胞癌(HCC)的重要危险因素。据世界卫生组织(WHO)统计,每年全球范围内约有4亿人受到HBV的慢性感染,而约有1亿人死于HBV感染。乙型肝炎病毒X蛋白(HBx)是由乙型肝炎病毒X基因编码的一个由154个氨基酸组成的多功能反式激活蛋白,在病毒的复制和肿瘤的形成和发展中发挥着极其重要的作用,大量研究表明HBx与肝细胞癌的发生、发展有着密切的关系。     

Replication of the hepatitis B virus requires a calcium-dependent HBx-induced G1 phase arrest of hepatocytes

Chronic HBV infections cause hepatocellular carcinoma (HCC). Activities of the HBV HBx protein regulate HBV replication and may contribute to the development of HCC. We previously reported that HBx causes primary rat hepatocytes to exit G0 but stall in G1 phase of the cell cycle; entry into G1 stimulated HBV replication. We now report that the activity of the mitochondria permeability transition pore is required for HBx regulation of cell cycle proteins and HBV replication in primary rat hepatocytes, that progression from G0 to G1 stimulates HBV polymerase activity, and that HBV replication is facilitated by the HBx-induced G1 arrest. HBx stimulation of HBV replication was linked to elevation of the R2 subunit of ribonucleotide reductase. Our studies suggest that HBx uses mitochondrial-dependent calcium signaling to cause hepatocytes to exit G0 but stall in G1 and that this HBx activity alters the cellular environment and stimulates HBV replication.     

Hepatitis B virus X protein upregulates expression of calpain small subunit 1 via nuclear facter‐κB/p65 in hepatoma cells

Hepatitis B virus (HBV) infection is closely correlated with the development of hepatocellular carcinoma (HCC), in which hepatitis B virus X protein (HBx) plays crucial roles. HBx is believed to be a multifunctional oncoprotein. It has been reported that the calpain small subunit 1 (Capn4) is upregulated in the HCC tissues and involved in the metastasis of HCC. Therefore, we suppose that HBx may promote hepatoma cell migration through Capn4. In the present study, we investigated the effect of HBx on regulating Capn4 expression in human HCC cells. Our data showed that HBx could increase promoter activity of Capn4 and upregulate the expression of Capn4 at the levels of mRNA and protein in human hepatoma HepG2 (or H7402) cells using luciferase reporter gene assay, real‐time quantitative RT‐PCR assay and Western blot analysis. While, the RNA interference targeting HBx mRNA was able to abolish the upregulation. Interestingly, we found that the inhibition of nuclear factor‐κB (NF‐κB) mediated by siRNA targeting NF‐κB/p65 mRNA or PDTC (an inhibitor of NF‐κB) could attenuate the upregulation of Capn4. While, HBx failed to increase the promoter activity of Capn4 in hepatoma cells when the putative NF‐κB binding site of the Capn4 promoter was mutant, suggesting that NF‐κB is involved in the activation of Capn4 mediated by HBx. In function, wound healing assay showed that HBx could significantly enhance the migration ability of HepG2 cells through upregulating Capn4. Thus, we conclude that HBx upregulate Capn4 through NF‐κB/p65 to promote migration of hepatoma cells. J. Med. Virol. 82:920–928, 2010. © 2010 Wiley‐Liss, Inc.     

乙型肝炎病毒HBs、HBx基因与HBVM的相关性及临床意义研究

目的：阐明HBs、HBx基因与HBVM的相关性及临床意义。方法：以HCCs、LCir、SeCH、CHB以及ASCs作为研究对象,应用ELISA法检测研究对象血清中HBsAg、HBsAb、HBeAg、HBeAb、HBcAb;应用PCR技术、探针杂交法及免疫化学自显影法定性检测HBs、HBx基因。结果：在研究对象中,HBsAg＋HBeAb＋HBcAb、HBsAg＋HBeAg＋HBcAb和HBsAg＋HBcAb模式阳性率分别为：53．3％、27．0％和11．8％;除ASCs组外,各临床类型肝病HBsAg＋HBeAb＋HBcAb模式阳性率均高于HBsAg＋HBeAg＋HBcAb模式的阳性率,但差别无统计学意义（P〉0．05）;与ASCs组比较,HCCs组的HBx基因阳性率明显高于ASCs组（P〈0．05）,其余各组均无明显差别（P〉0．05）。与ASCs组比较,LCir和SeCH组病人中的HBs基因阳性率显著高于ASCs组（P〈0．025）,其余各组无明显差异（P2〉0．05）;以HBsAg模式作为对照时,各HBVM模式的HBs、HBx基因阳性率与HBsAg模式的阳性率无明显差别（P〉0．05）;但在HBsAg＋HBeAg＋HBcAb模式中阳性率显著高于HBsAg＋HBeAb＋HBcAb模式的阳性率（P〈0．001）;HBsAg＋HBcAb模式中的HBx基因阳性率也显著高于HBsAg＋HBeAb＋HBcAb模式的阳性率（P〈0．001）。结论：HBsAg＋HBeAb＋HBcAb模式与肝病的发病关系似乎比HBsAg＋HBeAg＋HBcAb模式更为密切;HBx基因与HCCs的发生存在密切关系,而HBs基因与LCir、SeCH的发生关系密切。HBs、HBx基因与HBsAg＋HBeAg＋HBcAb模式关系密切,而HBx基因还与HBsAg＋HBcAb模式存在密切关系。     

HBx蛋白羧基端缺失对肝癌细胞生物学行为的影响

背景与目的:肝癌组织内普遍存在着截短表达的HBx蛋白,这种蛋白可能与肝癌的发生有关,本研究探讨截短表达的HBx蛋白和野生型HBx对人肝癌细胞Huh7生物学行为的影响。方法:脂质体法介导HBx突变体和野生型HBx重组体转染HBV(-)的Huh7细胞。PCR方法扩增Neo基因检测质粒DNA片段插入。通过MTT、平板克隆形成实验、流式细胞仪和裸鼠成瘤实验检测稳定转染细胞的生物学活性。结果:HBx3′-20和HBx3′-40组细胞生长速度较HBx3′-30组明显增快(P<0.05);HBx3′-20组[(17.34±2.77)%]和HBx3′-40组[(18.36±2.61)%]克隆形成率明显较HBx3′-30组[(7.31±1.44)%]和pcDNA3组[(6.87±2.38)%]高(P<0.05)。细胞周期检测结果显示,对比于野生型HBx组,HBx3′-20组和HBx3′-40组蛋白的表达能加速Huh7细胞由G0/G1期到S期的进程[S期:(36.96±1.82)%vs.(46.20±3.23)%,(53.99±4.02)%,P<0.05],相反,HBx3′-10和HBx3′-30组则出现G1期阻滞,而HBx组与pcDNA3空载体组间[(38.60±1.15)%]在S期无明显变化。裸鼠成瘤实验显示,HBx3′-40组[(3.19±0.34)cm3]成瘤体积明显大于HBx3′-30组[(1.58±0.27)cm3]、HBx组[(1.75±0.15)cm3]和pcDNA3组[(1.67±0.12)cm3],各组瘤重存在显著性差异(P<0.01)。结论:对比野生型HBx组,HBx3′-20和HBx3′-40对Huh7细胞的生长具有明显促增殖作用,HBx3′-30组显示出抑制作用。HBx通过缺失突变修饰其生物学功能,在原发性肝癌发生、发展过程中起着重要作用。     

Hepatitis B virus X protein promotes proliferation and upregulates TGF-β1 and CTGF in human hepatic stellate cell line,LX-2

BACKGROUND:Chronic hepatitis B virus(HBV)infection is a major cause of liver fibrosis,but the mechanisms underlying HBV-related fibrogenesis are still unknown. Although the roles of HBV X protein(HBx)remain poorly understood,it is thought to play an important role in the regulation of cellular growth and hepatocarcinogenesis. The aim of this study was to determine the role of HBx in liver fibrogenesis by studying the effect of HBx on the proliferation and expression of fibrosis-related molecules in the huma...