Hepatitis B virus X protein-mediated upregulation of miR-221 activates the CXCL12-CXCR4 axis to promote NKT cells in HBVrelated hepatocellular carcinoma

Both hepatitis B virus X protein(HBx)and microRNA-221(miR-221)have been implicated in the development of hepatitis B virus(HBV)-related hepatocellular carcinoma(HCC).The present study demonstrates that HBx promotes HCC cell proliferation via the C-X-C motif chemokine ligand 12-C-X-C chemokine receptor type 4(CXCL12-CXCR4)axis.We predict that HBx/miR-221-mediated CXCL12/CXCR4 signaling induces NKT cells to promote HBV-related HCC.Methods:After miR-221 mimic,miR-221 mimic negative control,miR-221 inhibitor,miR-221 inhibitor negative control were transfected into cells,the expression of CXCL12 and miR-221 was detected by qPCR and western blot.Then we constructed a stable HBV-HCC cell line.HBV-HCC cells were injected into the nude mice,thus a HBV-HCC mouse model was constructed.Q-PCR and western blot were used to detect the expression of HBx,miR-221,CXCL12 and CXCR4 in tumor tissues.The expression of CXCL12 was detected by immunohistochemistry,and the expression of CXCR4,CD3 and CD56 was detected by immunofluorescence.The levels of CXCL12,IL-2 and TNF-αin serum of mice were detected by ELISA.Sixty-one patients with HBV-related HCC,61 patients with HBV-related cirrhosis,61 patients with chronic hepatitis B(CHB)and 30 healthy people were enrolled.CXCL12,cytokine levels,and clinicopathological parameters were tested.Results:Hepatitis B virus X protein upregulates the expression of miR-221 and CXCL12 in lentivirus(LV5)-HBx-transfected HepG2 cells.HBx protein promotes HepG2 cell proliferation in vitro.HBx protein promoted tumor growth via the miR-221/CXCL12/CXCR4 pathway in a mouse tumor model.HBx protein upregulated natural killer T cell expression via the CXCR4/CXCL12 pathway to promote tumor growth.The data demonstrated a positive correlation between CXCL12 concentration with Cre levels and Child-Pugh scores.CXCL12 had an inferior diagnostic efficiency compared to IL-2 and IL-6 for HBV-related HCC.Conclusions:We present evidence that HBx/miR-221-mediated CXCL12/CXCR4 signaling induces NKT cells to promote HBV-related HCC.

Diagnostic value of gamma-glutamyltransferase/aspartate aminotransferase ratio, protein induced by vitamin K absence or antagonist II, and alpha-fetoprotein in hepatitis B virus-related hepatocellular carcinoma

BACKGROUND Researchers have investigated the diagnostic value of protein induced by vitamin K absence or antagonist II (PIVKA-II) and alpha-fetoprotein (AFP) in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC), and obtained abundant clinical diagnostic data. However, PIVKA-II and AFP have unsatisfactory specificity and sensitivity in the diagnosis of early-stage HBV-related HCC. Gamma-glutamyltransferase (γ-GT) and aspartate aminotransferase (AST) are common biomarkers for evaluating liver function, and we hypothesized that the γ-GT/AST ratio in combination with PIVKA-II and AFP would improve the diagnosis of early-stage HBV-related HCC. AIM To evaluate the diagnostic value of γ-GT/AST ratio alone or in combination with PIVKA-II and AFP in HBV-related HCC. METHODS Serum levels of γ-GT, AST, PIVKA-II, and AFP were detected and analysed in 176 patients with HBV-related HCC and in 359 patients with chronic hepatitis B. According to tumour size and serum level of HBV DNA, HBV-related HCC patients were divided into the following categories: Early-stage HCC patients, HCC patients, HBV DNA positive (HBV DNA+) HCC patients, and HBV DNA negative (HBV DNA-) HCC patients. Receiver-operating characteristic (ROC) curves were used to analyse and compare the diagnostic value of the single and combined detection of various biomarkers in different types of HBV-related HCC. RESULTS Tumour size was positively correlated with serum levels of PIVKA-II and AFP in HCC patients (r = 0.529, aP < 0.001 and r = 0.270, bP < 0.001, respectively), but there was no correlation between tumour size and the γ-GT/AST ratio (r = 0.073, P = 0.336). The areas under the receiver-operating characteristic curves (AUROCs) of the γ-GT/AST ratio in early-stage HCC patients, HBV DNA+ HCC patients and HBV DNA- HCC patients were not significantly different from that in the total HCC patients (0.754, 0.802, and 0.705 vs 0.779, respectively;P > 0.05). When PIVKA-II was combined with the γ-GT/AST ratio in the diagnosis of earlystage HCC, HCC, and HBV DNA+ HCC, the AUROCs of PIVKA-II increased, with values of 0.857 vs 0.835, 0.925 vs 0.913, and 0.958 vs 0.954, respectively. When AFP was combined with the γ-GT/AST ratio in the diagnosis of early-stage HCC, HCC, HBV DNA+ HCC, and HBV DNA- HCC, the AUROCs of AFP increased, with values of 0.757 vs 0.621, 0.837 vs 0.744, 0.868 vs 0.757, and 0.840 vs 0.828, respectively. CONCLUSION The γ-GT/AST ratio may be better than PIVKA-II and AFP in the diagnosis of early-stage HBV-related HCC, and its combination with PIVKA-II and AFP can improve the diagnostic value for HBV-related HCC.

Inhibition of apoptosis by oncogenic hepatitis B virus X protein: Implications for the treatment of hepatocellular carcinoma

Hepatitis B virus X protein(HBx) plays an important role in the development of hepatocellular carcinoma(HCC). In addition, hepatoma upregulated protein(HURP) is a cellular oncogene that is upregulated in a majority of HCC cases. We highlight here recent findings demonstrating a link between HBx, HURP and anti-apoptosis effects observed in cisplatin-treated HCC cells. We observed that Hep3B cells overexpressing HBx display increased HURP mRNA and protein levels, and show resistance to cisplatin-induced apoptosis. Knockdown of HURP in HBx-expressing cells reverses this effect, and sensitizes cells to cisplatin. The anti-apoptotic effect of HBx requires activation of the p38/MAPK pathway as well as expression of SATB1, survivin and HURP. Furthermore, silencing of HURP using short-hairpin RNA promotes accumulation of p53 and reduces cell proliferation in SK-Hep-1 cells(p53^(+/–)), whereas these effects are not observed in p53-mutant Mahlavu cells. Similarly, HURP silencing does not affect the proliferation of H1299 lung carcinoma cells or Hep3 B HCC cells which lack p53. Silencing of HURP sensitizes SK-Hep-1 cells to cisplatin. While HURP overexpression promotes p53 ubiquitination and degradation by the proteasome, HURP silencing reverses these effects. Inoculation of SK-Hep-1 cancer cells in which HURP has been silenced produces smaller tumors than control in nude mice. Besides, gankyrin, a positive regulator of the E3 ubiquitin ligase MDM2, is upregulated following HURP expression, and silencing of gankyrin reduces HURP-mediated downregulation of p53. In addition, we observed a positive correlation between HURP and gankyrin protein levels in HCC patients(r^2 = 0.778; n = 9). These findings suggest a role for the viral protein HBx and the host protein HURP in preventing p53-mediated apoptosis during cancer progression and establishment of chemoresistance.

Expression of HBx protein in hepatitis B virus-infected intrahepatic cholangiocarcinoma

BACKGROUND: Hepatitis B virus (HBV) is an etiological factor of intrahepatic cholangiocarcinoma (ICC), but the pathogenic mechanisms remain unclear. This study aimed to investigate the expression and possible role of HBx, an HBV- encoded potentially oncogenic protein, in HBV-infected ICC. METHODS: Tissue samples were obtained from 54 specimens of HBV-infected ICC. Forty-four specimens were of peripheral type and 10 hilar type. Formalin-fixed, paraffin-embedded sections of the specimens were immunohistochemically stained for HBx and p53. RESULTS: HBx expression was found in 70.4% (38/54) of the specimens, and it was more frequently seen in the peripheral type than in the hilar type (79.5% vs 30.0%, P=0.002). All three well-differentiated ICCs expressed HBx, whereas 76.9% (30/39) moderately-differentiated and 41.7% (5/12) poorly-differentiated ICCs had HBx expression (P=0.033). Patients with HBx expression had a significantly higher prevalence of elevated serum alpha-fetoprotein (P=0.033). p53 protein expression was found in 18 of 54 cases (33.3%), and was not correlated with that of HBx. CONCLUSIONS: HBx may contribute to the pathogenesis of ICC, particularly the peripheral type. p53 abnormality may not play a significant role in HBx-mediated oncogenicity during ICC carcinogenesis.

Virus entry mediated by hepatitis B virus envelope proteins

Hepatitis B virus(HBV),a major cause of human liver disease worldwide,encodes three envelope proteins needed for the attachment and entry of the virus into susceptible host cells.A second virus,hepatitis delta virus,which is known to enhance liver disease in HBV infected patients,diverts the same HBV envelope proteins to achieve its own assembly and infection.In the lab,lentiviral vectors based on human immunodeficiency virus type 1 can be assembled using the HBV envelope proteins,and will similarly infect susceptible cells.This article provides a partial review and some personal reflections of how these three viruses infect and of how recipient cells become susceptible,along with some consideration of questions that remain to be answered.

Hepatitis B virus X protein accelerates the development of hepatoma

The chronic infection of hepatitis B virus(HBV) is closely related to the occurrence and development of hepatocellular carcinoma(HCC). Accumulated evidence has shown that HBV X protein(HBx protein) is a multifunctional regulator with a crucial role in hepatocarcinogenesis. However, information on the mechanism by which HBV induces HCC is lacking. This review focuses on the pathological functions of HBx in HBV-induced hepatocarcinogenesis. As a transactivator, HBx can modulate nuclear factor kappa-light-chain-enhancer of activated B cells(NF-κB) and transcription factor AP-2. Moreover, HBx can affect regulatory non-coding RNAs(ncRNAs) including microRNAs and long ncRNAs(lncRNAs), such as miRNA-205 and highly upregulated in liver cancer(HULC), respectively. HBx is also involved in epigenetic modification, including methylation and acetylation. HBx interacts with various signal-transduction pathways, such as protein kinase B/Akt, Wnt/β-catenin, signal transducer and activator of transcription, and NF-κB pathways. Moreover, HBx affects cellular fate by shifting the balance toward cell survival. HBx may lead to the loss of apoptotic functions or directly contributes to oncogenesis by achieving transforming functions, which induce hepatocarcinogenesis. Additionally, HBx can modulate apoptosis and immune response by direct or indirect interaction with host factors. We conclude that HBx hastens the development of hepatoma.

Hepatitis B virus X protein promotes liver cell proliferation via a positive cascade loop involving arachidonic acid metabolism and p-ERK1/2

肝炎 B 病毒 X 蛋白质(HBx ) 在 hepatocellular 癌的发展起一个关键作用。这里，我们寻求了识别 HBx 由调停的机制肝房间增长。我们发现那 HBx upregulated cyclooxygenase-2 (COX-2 ) 的层次， 5-lipoxygenase (5 哈鱼) 和 phosphorylated 在肝房间的细胞外的调整信号的蛋白质 kinases 1/2 (p-ERK1/2 ) 。导致 HBx 的 p-ERK1/2 被 Gi/o 蛋白质，艇长或哈鱼的抑制废除。另外， HBx 增加了前列腺素 E2 (PGE2 ) 的数量，免除房间线的 leukotriene B4 (LTB4 ) 源于 hepatocytes。而且，这些释放 arachidonic 酸代谢物能激活 ERK1/2。有趣地，激活 ERK1/2 能 upregulate 以一种积极反馈方式的 COX-2 和 5 哈鱼的表示。在结论， HBx 经由包含 COX-2， 5 哈鱼，释放 arachidonic 酸代谢物， Gi/o 蛋白质和 p-ERK1/2 的一个积极反馈环提高并且维持肝房间增长。

Transactivating effect of complete S protein of hepatitis B virus and cloning of genes transactivated by complete S protein using suppression subtractive hybridization technique

AIM: To investigate the transactivating effect of complete S protein of hepatitis B virus (HBV) and to construct a subtractive cDNA library of genes transactivated by complete S protein of HBV by suppression subtractive hybridization (SSH) technique and to clone genes associated with its transactivation activity, and to pave the way for elucidating the pathogenesis of hepatitis B virus infection.METHODS: pcDNA3.1(-)-complete S containing full-length HBV S gene was constructed by insertion of HBV complete S gene into BarmH-I/Kpn I sites. HepG2 cells were cotransfected with pcDNA3.1(-)-complete S and pSV-lacZ.After 48 h, cells were collected and detected for the expression of β-galactosidase (β-gal). Suppression subtractive hybridization and bioinformatics techniques were used.The mRNA of HepG2 cells transfected with pcDNA3.1(-)-complete S and pcDNA3.1(-) empty vector was isolated,and detected for the expression of complete S protein by reverse transcription polymerase chain reaction (RT-PCR)method, and cDNA was synthesized. After digestion with restriction enzyme RcaI, cDNA fragments were obtained.Tester cDNA was then divided into two groups and ligated to the specific adaptors 1 and 2, respectively. After tester cDNA had been hybridized with driver cDNA twice and underwent nested PCR twice, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Amplification of the library was carried out within E. coli strain DH5α. The cDNA was sequenced and analyzed in GenBank with BLAST search after polymerase chain reaction (PCR) amplification.RESULTS: The complete S mRNA could be detected by RT-PCR in HepG2 cells transfected with the pcDNA3.1(-)-complete S. The activity of β-gal in HepG2 cells transfected with the pcDNA3.1(-)-complete S was 6.9 times higher than that of control plasmid. The subtractive library of genes transactivated by HBV complete S protein was constructed successfully. The amplified library contains 86 positive clones. Colony PCR showed that 86 clones contained DNA inserts of 200-1 000 bp, respectively.Sequence analysis was performed in 35 clones randomly,and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank, altogether 33 coding sequences were obtained. These cDNA sequences might be target genes transactivated by complete S protein of HBV. Moreover, two unknown genes were discovered, full length coding sequences were obtained by bioinformatics techniques,one of them was named complete S transactivated protein 1 (CSTP1) and registered in GenBank (AY553877).CONCLUSION: The complete S gene of HBV has a transactivating effect on SV40 early promoter. A subtractive cDNA library of genes transactivated by HBV complete S protein using SSH technique has been constructed successfully. The obtained sequences may be target genes transactivated by HBV complete S protein among which some genes coding proteins are involved in cell cycle regulation, metabolism, immunity, signal transduction, cell apoptosis and formation mechanism of hepatic carcinoma.

Hepatitis B virus X protein induces hepatic stem cell-like features in hepatocellular carcinoma by activating KDM5B

AIM To determine the role of hepatitis B virus X protein(HBx), HBx in regulating hepatic progenitor cell(HPC)-like features in hepatocellular carcinoma(HCC) and the underlying molecular mechanisms.METHODS We used a retrovirus vector to introduce wild type HBx or empty vector into Hep G2 cells. We then used these cells to analyze cell proliferation, senescence, transformation, and stem-like features. Gene expression profiling was carried out on Affymetrix GeneC hip Human U133A2.0 ver.2 arrays according to the manufacturer's protocol. Unsupervised hierarchical clustering analysis and Class Comparison analysis were performed by BRB-Array Tools software Version 4.2.2. A total of 238 hepatitis B virus(HBV)-related HCC patients' array data were used for analyzing clinical features.RESULTS The histone demethylase KDM5 B was significantlyhighly expressed in HBV-related HCC cases(P < 0.01). In HBV proteins, only HBx up-regulated KDM5 B by activating c-myc. Hepatic stem cell(Hp SC) markers(EpC AM, AFP, PROM1, and NANOG) were significantly highly expressed in KDM5B-high HCC cases(P < 0.01). KDM5 B played an important role in maintaining HpS Clike features and was associated with a poor prognosis. Moreover, inhibition of KDM5 B suppressed spheroid formation and cell invasion in vitro.CONCLUSION HBx activates the histone demethylase KDM5 B and induces HPC-like features in HCC. Histone demethylases KDM5 B may be an important therapeutic target against HBV-related HCC cases.

Inhibition of hepatitis B virus replication by pokeweed antiviral protein in vitro

AIM: To explore the inhibitory effects of pokeweed antiviral protein seed (PAP-S) and PAP encoded by a eukaryotic expression plasmid on hepatitis B virus (HBV) replication in vitro. METHODS: HepG2 2.2.15 cells in cultured medium were treated with different concentrations of PAP-S. HBsAg, HBeAg and HBV DNA in supernatants were determined by ELISA and fluorescent quantitative PCR respectively. MTT method was used to assay for cytotoxicity. HepG2 were cotransfected with various amounts of PAP encoded by a eukaryotic expression plasmid and replication competent wild-type HBV 1.3 fold over- length plasmid. On d 3 after transfection, HBsAg and HBeAg were determined by using ELISA. Levels of HBV core-associated DNA and RNA were detected by using Southern and Northern blot, respectively. RESULTS: The inhibitory effects of PAP-S on HBsAg, HBeAg and HBV DNA were gradually enhanced with the increase of PAP concentration. When the concentration of PAP-S was 10 μg/mL, the inhibition rates of HBsAg, HBeAg and HBV DNA were 20.9%, 30.2% and 50%, respectively. After transfection of 1.0 μg and 2.0 μg plasmid pXF3H-PAP, the levels of HBV nucleocapside- associated DNA were reduced by 38.0% and 74.0% respectively, the levels of HBsAg in the media by 76.8% and 99.7% respectively, and the levels of HBeAg by 72.7% and 99.3% respectively as compared with controls. Transfection with 2 μg plasmid pXF3H-PAP reduced the levels of HBV nucleocapside-associated RNA by 69.0%.CONCLUSION: Both PAP-S and PAP encoded by a eukaryotic expression plasmid could effectively inhibit HBV replication and antigen expression in vitro, and the inhibitory effects were dose-dependent.

Targeting hepatitis B virus antigens to dendritic cells by heat shock protein to improve DNA vaccine potency

AIM: To investigate a novel DNA vaccination based upon expression of the HBV e antigen fused to a heat shock protein (HSP) as a strategy to enhance DNA vaccine potency. METHODS: A pCMV-HBeAg-HSP DNA vaccine and a control DNA vaccine were generated. Mice were immunized with these different construct. Immune responses were measured 2 wk after a second immunization by a T cell response assay, CTL cytotoxicity assay, and an antibody assay in C57BL/6 and BALB/c mice. CT26-HBeAg tumor cell challenge test in vivo was performed in BALB/c mice to monitor anti-tumor immune responses. RESULTS: In the mice immunized with pCMV-HBe-HSP DNA, superior CTL activity to target HBV-positive target cells was observed in comparison with mice immunized with pCMV-HBeAg (44% ± 5% vs 30% ± 6% in E: T > 50:1, P < 0.05). ELISPOT assays showed a stronger T-cell response from mice immunized with pCMV-HBe-HSP than that from pCMV-HBeAg immunized animals when stimulated either with MHC classⅠor class Ⅱ epitopes derived from HBeAg (74% ± 9% vs 31% ± 6%, P < 0.01). ELISA assays revealed an enhanced HBeAg antibody response from mice immunized with pCMV-HBe-HSP than from those immunized with pCMV-HBeAg. The lowest tumor incidence and the slowest tumor growth were observed in mice immunized with pCMV-HBe-HSP when challenged with CT26-HBeAg.CONCLUSION: The results of this study demonstrate a broad enhancement of antigen-specific CD4+ helper,CD8+ cytotoxic T-cell, and B-cell responses by a novel DNA vaccination strategy. They also proved a stronger antigen-specific immune memory, which may be superior to currently described HBV DNA vaccination strategies for the treatment of chronic HBV infection.

Screening and identification of interacting proteins with hepatitis B virus core protein in leukocytes and cloning of new gene C1

瞄准：在外部血在 HBV 复制的致病调查 HBcAg 的生物功能单音的原子房间(PBMC ) 。方法：HBcAg 区域被聚合酶链反应(PCR ) 放大， HBV HBcAg 诱饵原生质标志 pGBKT7-HBcAg 被平淡的分子的生物方法构造。然后， recombinant 原生质标志 DNA 被转变成酵母 AH109。在 HBV 核心蛋白质在 AH109 酵母紧张(西方的污点分析) 被表示以后，酵母 -- 屏蔽的二个混血儿被与包含白血球 cDNA 图书馆的 Y187 交配 AH109 执行原生质标志。双酵母房间是合成退学学生营养培养基(SD/-Trp-Leu-His-Ade )(QDO ) 和合成退学学生营养培养基(SD/-Trp-Leu-His-Ade )(TDO ) 上的 plated。第二屏蔽与 LacZ 报告基因被执行(酵母房间在 QDO 被种中等包含 X-alpha-gal ) 。在从积极殖民地获得的 HBV 核心蛋白质和蛋白质之间的相互作用被重复酵母进一步证实 -- 二个混血儿。在原生质标志 DNA 从蓝殖民地被提取并且定序以后，结果被生物信息的方法分析。结果：十八个殖民地被获得并且定序，包括在癌症 2 的亢奋的甲基化( 3 关口)，真核细胞的翻译延伸因素 2 ( 2 关口)，乙酰辅酶 A 合成酶 3 ( 1 个关口一个)， DNA 聚合酶鲸鱼群妈( 1 个关口一个)，通常认为的翻译开始因素( 1 个关口一个)， chemokine ( C-C 主题)受体 5 ( 1 个关口一个)， mitochondrial ribosomal 蛋白质 L41 ( 1 个关口一个)， kyot 绑定蛋白质基因( 1 个关口一个)， RanBPM ( 1 个关口一个)，HBeAg有约束力的蛋白质 3 ( 1 个关口一个)，规划了细胞死亡 2 ( 1 个关口一个)。有未知功能的四新基因被识别。结论：在白血球的核心蛋白质交往蛋白质可以提供的 HBV 的基因的成功的克隆为学习 HBV 的生物功能的一些新线索核心蛋白质。

Polymorphisms of microsomal triglyceride transfer protein in different hepatitis B virus-infected patients

AIM: To identify the two polymorphisms of microsomal triglyceride transfer protein (MTP) gene in the Chinese population and to explore their correlation with both hepatitis B virus (HBV) self-limited infection and persistent infection. METHODS: A total of 316 subjects with self-limited HBV infection and 316 patients with persistent HBV infection (195 subjects without familial history), matched with age and sex, from the Chinese Han population were enrolled in this study. Polymorphisms of MTP at the promoter region -493 and at H297Q were determined by the allele specific polymerase chain reaction (PCR). RESULTS: The ratio of males to females was 2.13:1 for each group and the average age in the self-limited and chronic infection groups was 38.36 and 38.28 years, respectively. None of the allelic distributions deviated significantly from that predicted by the Hardy-Weinberg equilibrium. There was a linkagedisequilibrium between H297Q and -493G/T (D’ = 0.77). As the χ2 test was used, the genotype distribution of MTP -493G/T demonstrated a significant difference between the self-limited infection group and the entire chronic group or the chronic patients with no family history (χ2 = 8.543, P = 0.015 and χ2 = 7.199, P = 0.019). The allele distribution at the MTP-493 position also demonstrated a significant difference between the study groups without family history (χ2 = 6.212, P = 0.013). The T allele emerged as a possible protective factor which may influence the outcomes of HBV infection (OR: 0.59; 95% CI: 0.389-0.897). CONCLUSION: The polymorphism of the MTP gene, T allele at -493, may be involved in determining the HBV infection outcomes, of which the mechanism needs to be further investigated.

Novel Evidence Suggests Hepatitis B Virus Surface Proteins Participate in Regulation of HBV Genome Replication

Naturally occurring mutations in surface proteins of Hepatitis B virus(HBV) usually result in altered hepatitis B surface antigen(HBsAg) secretion efficiency.In the present study,we reported two conserved residues,M75 and M103 with respect to HBsAg,mutations of which not only attenuated HBsAg secretion(M75 only),but also suppressed HBV genome replication without compromising the overlapping p-gene product.We also found M75 and M103 can initiate truncated surface protein(TSPs) synthesis upon over-expression of full-length surface proteins,which may possibly contribute to HBV genome replication.However,attempts to rescue replicationdefective HBV mutant by co-expression of TSPs initiated from M75 or M103 were unsuccessful,which indicated surface proteins rather than the putative TSPs were involved in regulation of HBV genome replication.

Interferon-alpha restrains growth and invasive potential of hepatocellular carcinoma induced by hepatitis B virus X protein

AIM: To investigate the effects of interferon-alpha (IFN-α) to restrain the growth and invasive potential of hepatocellular carcinoma (HCC) induced by hepatitis B virus (HBV) X protein. METHODS: The pcDNA3.1-HBx plasmid was transfected into Chang cells by Lipofectamine in vitro, and Chang/HBx was co-cultured with IFN-α. Cell survival growth curve and clonogenicity assay were used to test the growth potential of Chang/pcDNA3.1, Chang/HBx and IFN-α-Chang/HBx in vitro. Growth assay in nude mice was used to detect the growth potential of Chang/ pcDNA3.1, Chang/HBx and IFN-α-Chang/HBx in vivo. Wound healing and transwell migration assays were used to detect the invasive ability of Chang/pcDNA3.1, Chang/HBx and IFN-α-Chang/HBx. RESULTS: Compared with CCL13 cells transfected with pcDNA3.1, CCL13 with stable expression of hepatitis B virus X protein showed the characteristics of malignant cells with high capability of growth and invasion by detecting their growth curves, colony forming efficiency, wound healing , transwell migration assays and growth assays in nude mice. Its capability of growth and invasion could be controlled by IFN-α. CONCLUSION: IFN-α can restrain the growth and invasive potential of HCC cells induced by HBx protein, which has provided an experimental basis for IFN-α therapy of HCC.

Hepatitis B virus X protein regulates the mEZH2 promoter via the E2F1-binding site in AML12 cells

Histone lysine methyltransferase EZH2 has been reported to be frequently overexpressed in hepatocellular carcinoma(HCC) tissues and associated with hepatocarcinogenesis.However,the exact mechanism of EZH2 up-regulation in HCC has not been determined.In this study,we used murine hepatocyte AML12 cells to investigate the role of hepatitis B virus X protein(HBx) in regulating the expression of mEZH2.Western blot analysis demonstrated that the expression level of mEZH2 protein in AML12 cells was up-regulated by HBx in a dose-dependent manner.To further investigate the mechanism of mEZH2 overexpression,the 2500 bp regulatory sequence upstream from the first exon of the mEZH2 gene was amplified from AML12 genomic DNA and constructed into a luciferase reporter plasmid.The luciferase activity of the mEZH2 promoter significantly increased in AML12 cells co-transfected with HBx plasmid,and deleting the-486/-214 promoter region decreased HBx-induced mEZH2 promoter activation by nearly 50%.The-486/-214 region was then analyzed in the TRANSFAC 6.0 database and a typical E2F1-binding site was found.Mutation of this E2F1-binding site or knockdown of E2F1 expression by RNAi led to a dramatic decrease in HBx-induced activation of the mEZH2 promoter and mEZH2 overexpression in AML12 cells.These results provide evidence that HBx up-regulates mEZH2 expression by transactivating the mEZH2 promoter through E2F1 transcription factor,thereby providing new epigenetic evidence for the carcinogenic effect of HBx.

Antiviral Effect of Interferon-Induced Guanylate Binding Protein-1 against Coxsackie Virus and Hepatitis B Virus B3 in Vitro

Guanylate binding protein-1(GBP-1)is an interferon-induced protein.To observe its antiviral effect against Hepatitis B virus(HBV)and Coxsackie virus B3(CVB3),we constructed an eukaryotic expression vector of human GBP-1(hGBP-1).Full-length encoding sequence of hGBP-1 was amplified by long chain RT-PCR and inserted into a pCR2.1 vector,then subcloned into a pCDNA3.1(-)vector.Recombinant hGBP-1 plasmids and pHBV1.3 carrying 1.3-fold genome of HBV were contransfected into HepG2 cells,and inhibition effect of hGBP-1 against HBV replication was observed.Hela cells transfected with recombinant hGBP-1 plasmids were challenged with CVB3,and viral yield in cultures were detected.The results indicated that recombinant eukaryotic expression plasmid of hGBP-1 was constructed successfully and the hGBP-1 gene carried in this plasmid could be efficiently expressed in HepG2 cells and Hela cells.hGBP-1 inhibit CVB3 but not HBV replication in vitro.These results demonstrate that hGBP-1 mediates an antiviral effect against CVB3 but not HBV and perhaps plays an important role in the interferon-mediated antiviral response against CVB3.

Screening of hepatocyte proteins binding to complete S protein of hepatitis B virus by yeast-two hybrid system

AIM: To investigate the biological function of complete S protein and to look for proteins interacting with complete S protein in hepatocytes.METHODS: We constructed bait plasmid expressing complete S protein of HBV by cloning the gene of complete S protein into pGBKT7, then the recombinant plasmid DNA was transformed into yeast AH109 (a type). The transformed yeast AH109 was mated with yeast Y187 (α type) containing liver cDNA library plasmid in 2xYPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/Trp-Leu-His-Ade) containing X-α-gal for selection and screening. After extracting and sequencing of plasmids from positive (blue) colonies, we underwent sequence analysis by bioinformatics.RESULTS: Nineteen colonies were selected and sequenced.Among them, five colonies were Homo sapiens solute carrier family 25, member 23 (SLC25A23), one was Homo sapiens calreticulin, one was human serum albumin (ALB)gene, one was Homo sapiens metallothionein 2A, two were Homo sapiens betaine-homocysteine methyltransferase,three were Homo sapiensNa+ and H+ coupled amino acid transport system N, one was Homo sapiens CD81 antigen (target of anti-proliferative antibody 1) (CD81), three were Homo sapiens diazepam binding inhibitor, two colonies were new genes with unknown function.CONCLUSION: The yeast-two hybrid system is an effective method for identifying hepatocyte proteins interacting with complete S protein of HBV. The complete S protein may bind to different proteins i.e., its multiple functions in vivo.

Autophagy related protein 9A increase in hepatitis B virusassociated hepatocellular carcinoma and the role in apoptosis

The majority of hepatocellular carcinoma(HCC)cases are associated with the hepatitis B virus(HBV)infection.Autophagy related protein 9A(ATG9A)is a transmembrane protein required for autophagosome formation.In order to investigate the role of ATG9A in HBV-associated HCC,ATG9A protein expression was determined in tumor liver tissues and compared with adjacent nontumor tissues from HCC patients with or without HBV infection.In HBVassociated HCC tissues,ATG9A protein level was increased in tumor liver tissues,but not in cases of non-HBV HCC.Our findings suggested that ATG9A might be involved in HBV and cancer cell survival.Therefore,we aimed to analyze the function of ATG9A in HBV replication using RNA interference to evaluate the HBV DNA level using real-time PCR.In the present study,there were no significant differences between shATG9A-transfected HepG2.2.15 cells and the mock control.However,we found that silencing ATG9A affected apoptosis in HepG2.2.15 and HepG2 cell lines.Our results indicated that ATG9A might be partly involved in the survival of HCC.Thus,the inhibition of ATG9A together with other targets might be a potential drug target for HCC treatment.

Screening and cloning for proteins transactivated by the PS1TP5 protein of hepatitis B virus:A suppression subtractive hybridization study

AIM:To clone and identify human genes transactivated by PS1TP5 by constructing a cDNA subtractive library with suppression subtractive hybridization(SSH)technique.METHODS:SSH and bioinformatics techniques were used for screening and cloning of the target genes transactivated by PS1TP5 protein.The mRNA was isolated from HepG2 cells transfected with pcDNA3.1(-)-myc-his(A)-PS1TP5 and pcDNA3.1(-)-myc-his(A)empty vector,respectively,and SSH technique was employed to analyze the differentially expressed DNA sequence between the two groups.After digestion with restriction enzyme RsaⅠ,small size cDNAs were obtained.Then tester cDNA was divided into two groups and ligated to the specific adaptor 1 and adaptor 2,respectively.The tester cDNA was hybridized with driver cDNA twice and subjected to nested PCR for two times,and then subcloned into T/A plasmid vectors to set up the subtractive library.Amplification of the library was carried out with E.coli strain DH5α.The cDNA was sequenced and analyzed in GenBank with Vector NTI 9.1 and NCBI BLAST software after PCR amplification.RESULTS:The subtractive library of genes transactivated by PS1TP5 was constructed successfully.The amplified library contained 90 positive clones.Colony PCR showed that 70 clones contained 200-1000-bp inserts.Sequence analysis was performed in 30 clones randomly,and the full-length sequences were obtained by bioinformatics technique.Altogether 24 coding sequences were obtained,which consisted of 23 known and 1 unknown.One novel gene with unknown functions was found and named as PS1TP5TP1 after being electronically spliced,and deposited in GenBank(accession number:DQ487761).CONCLUSION:PS1TP5 is closely correlated with immunoregulation,carbohydrate metabolism,signal transduction,formation mechanism of hepatic fibrosis,and occurrence and development of tumor.Understanding PS1TP5 transactive proteins may help to bring some new clues for further studying the biological functions of pre-S1 protein.