HBx、MHBs~（t155）真核表达载体构建及在HepG2细胞中的表达

目的建立稳定、高效的HBx、羧基末端截短的中分子表面蛋白（MHBst）体外表达细胞株,以进一步研究HBx、MHBst蛋白在肝癌发生中的作用及其机理。方法设计特异性引物,采用PCR方法从adr亚型HBV质粒pHBV DNA中扩增HBx、羧基末端截短至155位氨基酸的中分子表面蛋白（MHBst155）编码基因,并定向插入绿色荧光蛋白（GFP）表达载体pEGFP-C1的BglⅡ、KpnⅠ和BglⅡ、BamHⅠ酶切位点,转化宿主菌DH5α,提取质粒,分别用上述内切酶酶切及DNA测序鉴定重组质粒。采用脂质体介导将空载体、重组质粒分别转染到人肝肿瘤细胞株HepG2中,G418筛选抗性细胞克隆,荧光显微镜下观察GFP表达,挑取表达GFP的抗性克隆扩大培养、传代。采用RT-PCR、蛋白印迹检测抗性细胞中HBx、MHBst155的mRNA及蛋白水平。结果经酶切及测序鉴定成功构建了pGFP-HBx、pGFP-MHBst155重组表达载体,将空载体及重组质粒转染HepG2,经G418筛选约20 d获得抗性细胞克隆。将带有绿色荧光的抗性克隆扩大培养并经传代40次,细胞仍表达强的绿色荧光。RT-PCR及蛋白印迹检测表明转染重组体的HepG2/GFP-HBx、HepG2/GFP-MHBst155细胞有相应的条带,而空载体、空白对照组未出现条带。结论成功构建了HBx、MHBst155真核重组表达载体pGFP-HBx、pGFP-MHBst155,获得了稳定表达融合蛋白GFP-HBx、GFP-MHBst155的HepG2细胞系,为进一步研究HBx及MHBst蛋白在肝癌发生中的作用及分子机制构建了良好的平台。     

乙型肝炎病毒X蛋白通过降低P4启动子甲基化水平上调人IGF-II基因的转录

目的:构建稳定表达乙型肝炎病毒X蛋白(HBx)的肝癌细胞株HepG2-HBx,探讨HBx对胰岛素样生长因子 II(IGF-II)基因P4启动子甲基化水平及转录表达的影响。方法:应用基因重组技术,构建含HBx基因的重组逆转录病毒载体pBABE-puro-HBx,采用磷酸钙共沉淀法将其转染293FT包装细胞产生逆转录病毒,感染HepG2肝癌细胞,采用嘌呤霉素进行阳性克隆筛选,Western blotting鉴定表达HBx蛋白的肝癌细胞株HepG2-HBx。采用亚硫酸氢盐测序法及实时荧光定量RT-PCR检测HepG2-HBx细胞中P4启动子甲基化水平及P4 mRNA表达水平变化。进一步将体外甲基化的人IGF-II基因P4启动子驱动的荧光素酶报告载体pGL3-P4及含HBx基因的pCMV-tag2B-X质粒共转染HepG2肝癌细胞,采用亚硫酸氢盐测序法及双萤光素酶实验检测pGL3-P4载体上P4启动子甲基化水平及转录调控活性变化。结果:(1)经Western blotting鉴定,成功构建了稳定表达HBx蛋白的肝癌细胞株HepG2-HBx;(2)表达HBx蛋白的HepG2-HBx细胞中P4启动子甲基化CpG位点的比例(9.0%)明显低于对照细胞HepG2-control(25.0%)(P<0.01),而其P4 mRNA表达水平则为对照细胞HepG2-control的2.8倍;(3)共转染pCMV-tag2B-X质粒的HepG2细胞中pGL3-P4载体上P4启动子甲基化CpG位点的比例(60.8%)明显低于共转染对照质粒pCMV-tag2B的HepG2细胞(84.1%)(P<0.01),而前者P4启动子相对萤光素酶活性(14.12±0.89)则明显高于后者(4.61±0.76)(P<0.01)。结论:HBx蛋白可降低IGF-II基因P4启动子甲基化水平,进而上调其转录表达。     

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.     

Hepatitis B virus regulates Raf1 expression in HepG2.2.15 cells by enhancing its promoter activity

Raf1 kinase is a central component of many signaling pathways that are involved in normal cell growth and oncogenic transformation. The expression of Raf1 is significantly increased in hepatocellular carcinoma (HCC). HBV is a major risk factor for HCC. HBx protein can increase the expression of Raf1; however, the mechanism of how HBV regulates Raf1 expression is still unknown. In this study, we showed the Raf1 expression was significantly higher in HepG2.2.15 cells than that in HepG2 cells in vitro. HBV could up-regulate Raf1 expression by enhancing the activity of its promoter in a dose-dependent manner, and HBs and HBx may be involved in this process. After silencing HBs and HBx by using RNA interference, the expression of Raf1 in HepG2.2.15 cells could be significantly inhibited. These results might provide useful information for understanding the mechanism of HCC induced by HBV infection.     

Adiponectin, a downstream target gene of peroxisome proliferator-activated receptor γ, controls hepatitis B virus replication

In this study, HepG2-hepatitis B virus (HBV)-stable cells that did not overexpress HBx and HBx-deficient mutant-transfected cells were analyzed for their expression of HBV-induced, upregulated adipogenic and lipogenic genes. The mRNAs of CCAAT enhancer binding protein α (C/EBPα), peroxisome proliferator-activated receptor γ (PPARγ), adiponectin, liver X receptor α (LXRα), sterol regulatory element binding protein 1c (SREBP1c), and fatty acid synthase (FAS) were expressed at higher levels in HepG2-HBV and lamivudine-treated stable cells and HBx-deficient mutant-transfected cells than in the HepG2 cells. Lamivudine treatment reduced the mRNA levels of PPARγ and C/EBPα. Conversely, HBV replication was upregulated by adiponectin and PPARγ agonist rosiglitazone treatments and was downregulated by adiponectin siRNAs. Collectively, our results demonstrate that HBV replication and/or protein expression, even in the absence of HBx, upregulated adipogenic or lipogenic genes, and that the control of adiponectin might prove useful as a therapeutic modality for the treatment of chronic hepatitis B.     

Involvement of mitochondrial permeability transition in hepatitis B virus replication

The HBx protein of human hepatitis B virus (HBV) activates a calcium-dependent kinase pathway which is essential for the viral replication. In this study, we found that HBx expression in the absence of other HBV proteins and in the context of HBV replication decreased the mitochondrial calcein-AM/CoCl₂ signals by 10% and 14% in HepG2 cells and by 15% and 10% in Huh7 cells, respectively. This indicates that HBx can induce mitochondrial permeability transition (MPT) and cause calcium effusion into the plasma. In addition, RNA interference of Cylophilin D decreased HBx-induced MPT and suppressed HBV DNA replication by 41% in HepG2 cells. Our results suggest that HBx expression can induce MPT and facilitate HBV DNA replication.     

The expression and role of lncRNA AX800134 in hepatitis B virus-related hepatocellular carcinoma

Chronic infection with hepatitis B virus (HBV) is one of most important risk factors for the development of hepatocellular carcinoma (HCC). Several long non-coding RNAs (lncRNAs) have been shown to be involved in the etiology of HBV-related HCC. AX800134 is one recently identified lncRNA associated with HCC. In this study, we validated the upregulated expression of AX800134 in HBV-positive HCC compared with HBV-negative HCC. Furthermore, we found that HBV X protein (HBx) directly triggered AX800134 expression in human hepatoma HepG2 cells. Pro-inflammatory cytokine TNFα also induced AX800134 upregulation in HBx-expressing HepG2 cells, which could be reversed by reactive oxygen species (ROS) scavenger pyrrolidine dithiocarbamate (PDTC). Additionally, silencing AX800134 with siRNA interference remarkably inhibited the growth and invasion of HBx-expressing HepG2 cells. AX800134 antagonism also enhanced spontaneous apoptosis of HepG2 cells under serum deprivation condition. Therefore, our results indicate that highly expressed AX800134 acts as an oncogenic factor in HCC, and its upregulation is related with the viral product HBx and chronic inflammation.     

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.     

Calcium ions affect the hepatitis B virus core assembly

Previous report showed that cytosolic Ca2+ induced by hepatitis B virus X protein (HBx) promotes HBV replication. In this study, in vitro experiments showed that (i) HBV core assembly in vitro was promoted by Ca2+ through the sucrose density gradient and the analytical ultracentrifuge analysis. Also, (ii) transmission electron microscope analysis demonstrated these assembled HBV core particles were the capsids. Ex vivo experiments showed that the treatment of BAPTA-AM and cyclosporine A (CsA) reduced HBV capsids in the transfected HepG2 cells. In addition to that, the treatment of Thapsigargin (TG) increased HBV capsids in the transfected HepG2 cells. Furthermore, we investigated the increased HBV core assembly by HBx. The results show that the increased cytosolic calcium ions by HBx promote the HBV core assembly.     

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.     

HBV preS2S-rhGM-CSF融合基因表达质粒的构建和表达

目的：研究GM-CSF和preS2对乙肝DNA疫苗的免疫增强作用。方法：采用PCR方法，扩增HBV preS2 S基因约846 bp的片段和rhGM-CSF(包括甘氨酸接头)基因450bp的片段。通过T-A克隆技术和基因定向克隆，构建融合基因的真核表达质粒pcDNA3．1-S2S-rhGM-CSF，并在HepG2细胞中表达。结果：经酶切、PCR及DNA测序鉴定，融合基因表达质粒HBV preS2S-rhGM-CSF成功地构建。将其转染HepG2细胞后，目的基因的转录通过RT-PCR得到证实，而且表达的融合蛋白能与抗-HBs、抗-preS2和抗-GM-CSF单克隆抗体(mAb)均产生特异性反应。结论：融合基因表达载体pcDNA3．1-S2S-rhGM-CSF的成功构建并表达，为进一步研究乙肝DNA疫苗奠定了实验基础。     

Hepatitis B virus regulatory HBx protein binding to DDB1 is required but is not sufficient for maximal HBV replication

Robust hepatitis B virus (HBV) replication is stimulated by the regulatory HBx protein. HBx binds the cellular protein DDB1; however, the importance of this interaction for HBV replication remains unknown. We tested whether HBx binding to DDB1 was required for HBV replication using a plasmid based replication assay in HepG2 cells. Three DDB1 binding-deficient HBx point mutants (HBx⁶⁹, HBx^90/91, HBx^R96E) failed to restore wildtype levels of replication from an HBx-deficient plasmid, which established the importance of the HBx-DDB1 interaction for maximal HBV replication. Analysis of overlapping HBx truncation mutants revealed that both the HBx-DDB1 binding domain and the carboxyl region are required for maximal HBV replication both in vitro and in vivo, suggesting the HBx-DDB1 interaction recruits regulatory functions critical for replication. Finally we demonstrate that HBx localizes to the Cul4A-DDB1 complex, and discuss the possible implications for models of HBV replication.     

The C-terminal region of the hepatitis B virus X protein is required for its stimulation of HBV replication in primary mouse hepatocytes

Hepatitis B virus (HBV) infection is an important risk factor for hepatocellular carcinoma (HCC). The hepatitis B virus X protein (HBx), a multifunctional regulatory protein encoded by HBV, is known to be involved in stimulation of viral replication by modulating cell cycle status. HBx is required for maximal virus replication in plasmid-based replication assays in immortalized human liver HepG2 cells and in primary rat hepatocytes. Moreover, the C-terminal region of HBx is important for HBV replication in HepG2 cells. However, in normal hepatocytes, the region of HBx that is responsible for its effect on cell cycle regulation and HBV replication is unclear. We have demonstrated that HBx is similarly required for maximal HBV replication in primary mouse hepatocytes and that the C-terminus of HBx is essential for its ability to stimulate HBV replication by inducing quiescent hepatocytes to exit G0 phase of the cell cycle but stall in G1 phase. Our studies establish that primary mouse hepatocytes support HBx-dependent HBV replication, and provide further evidence for the effect of the C-terminal region of HBx on HBV infection and replication.     

Interleukin-32 expression induced by hepatitis B virus protein X is mediated through activation of NF-κB

HBV replicates noncytopathically in hepatocytes, but HBV or proteins encoded by HBV genome could induce cytokines, chemokines expression by hepatocytes. Moreover, liver damage in patients with HBV infection is immune-mediated and cytokines play important roles in immune-mediated liver damage after HBV infection. Interleukin-32 (IL-32) is a proinflammatory cytokine and plays a critical role in inflammation. However, the role of HBV in IL-32 expression remains unclear. In the present study, we demonstrate that hepatitis B virus protein X (HBx) increases IL-32 expression through the promoter of IL-32 at positions from -746 to +25 and in a dose-dependent manner. Furthermore, we demonstrate that increase of NF-κB subunits p65 and p50 in Huh7 cells also augments IL-32 expression, and the NF-κB inhibitor blocks the effect of HBx on IL-32 induction. These results indicate that NF-κB activation is required for HBx-induced IL-32 expression. In conclusion, IL-32 is induced by HBx in Huh7 cells. Our results suggest that IL-32 might play an important role in inflammatory response after HBV infection.     

A broadly reactive monoclonal antibody detects multiple genotypes of hepatitis B virus X protein

A highly specific and broadly reactive monoclonal antibody against hepatitis B virus X (HBx) protein was developed that detected, in both immunoblotting and immunofluorescence, HBx proteins of seven of the eight currently known genotypes of HBV, which were overexpressed in cultured cells. Evaluation of HBx expression levels in cultured hepatocytes using this monoclonal antibody showed that cells transiently and stably transfected with HBV genomes expressed far less HBx protein than cells transiently transfected with an HBx overexpression plasmid routinely used for studying HBx functions. The availability of such sensitive and broadly reactive monoclonal antibodies against HBx will enable more-quantitative studies of HBx functions.     

乙型肝炎病毒X蛋白对肝癌细胞生长的影响

目的探讨乙型肝炎病毒X蛋白（hepatitis Bvirus Xprotein，HBx）对肝癌细胞恶性程度的影响。方法构建携带HBV。基因真核表达载体pcDNA用Bx，以脂质体介导转染HepG2肝癌细胞，建立可稳定表达HBx的肝癌细胞系HepG2-HBx细胞，同时设空载体pcDNA，转染细胞HepG2-pcDNA，及未转染HepG2细胞为对照组。PCR法扩增Neo基因检测插入的质粒DNA片段．免疫荧光检测HBx的表达。通过生长曲线测定、平板克隆形成实验、MTr比色实验、Hoechst33342核形态学染色观察及流式细胞仪测定．了解稳定转染细胞的生物学行为变化。结果与对照组相比，转染pcDNA√IBx的HepG2-HBx细胞生长速度加快．其倍增时间缩短（28h对32．5h或34h，P〈0．05），克隆形成率增加[（10．12±0．23）％对（5．33±O．19）％或（5．19±0．28）％，P〈0．05]，细胞周期分析显示由GdG，期-S期的进程明显加快。细胞凋亡检测显示HepG2-HB。细胞可抵抗放线菌素D（ActD）诱导的凋亡作用。结论HBx可提高肝癌细胞的增殖活性，并增强肝癌细胞的抗凋亡能力．增加了肝癌细胞的恶性表型。