Progress in the role of ubiquitin-proteasome system in hepatitis B virus X protein-mediated HBV infection and hepatocarcinogenesis / 中华微生物学和免疫学杂志

Chronic hepatitis B virus (HBV) infection is one of the leading causes of hepatocellular carcinoma (HCC). Ubiquitin-proteasome system (UPS) mediates the post-translational modification and degradation of protein in eukaryotic cells. Recent studies have revealed that UPS plays an important role in HBV infection and hepatocarcinogenesis. Targeting HBx to intervene in the progression of HBV infection or HBV-related HCC has become a research hotspot both domestically and internationally in recent years. This article reviewed the progress in HBx promoting HBV infection and hepatocarcinogenesis through UPS.

Protein phosphatase 4 catalytic subunit (PP4C) increases hepatitis B virus X protein levels and promotes its biological functions / 中华微生物学和免疫学杂志

Objective:To investigate the role of protein phosphatase 4 catalytic subunit (PP4C) in regulating hepatitis B virus X protein (HBx) levels and its effects on the biological functions of HBx, thus to provide a potential therapeutic targets for hepatitis B virus (HBV)-related hepatocellular carcinoma.Methods:In vivo and in vitro interactions between HBx and PP4C were analyzed by co-immunoprecipitation (Co-IP) and GST pull-down assay. Recombinant plasmids of PP4C and HBx were co-transfected with Lipofectamine 3000 reagents into hepatoma cells to detect the protein levels of HBx by Western blot. The half-life of HBx in the transfected cells treated with cycloheximide (CHX) were detected. The phosphorylation assay was used to evaluate the effects of PP4C on HBx phosphorylation. CCK8 assay, wound healing assay and Matrigel invasion chamber assay were used to analyze the effects of PP4C on the biological functions of HBx. Results:PP4C interacted with HBx in vivo and in vitro. PP4C overexpression significantly increased the protein level and stability of HBx and the phosphorylation assay confirmed that PP4C overexpression decreased the serine phosphorylation of HBx in hepatoma cells. PP4C overexpression enhanced the migration and invasion of hepatoma cells, but had no significant effects on the proliferation. Conclusions:The interactions between HBx and PP4C promoted the stability of HBx and ultimately enhanced the migration and invasion of hepatoma cells, and the mechanisms might be related to the decrease of HBx serine phosphorylation by PP4C. This study provided a theoretical basis for further investigation of the pathogenic mechanisms of HBx, and targeting PP4C and HBx interaction might provide insights for developing novel treatment for HBV-related hepatocellular carcinoma.

Effect and significance of viral proteins regulating activity of retroelement long interspersed element 1 / 中国生物制品学杂志

@#Abstract：Long interspersed element⁃1（LINE⁃1）is the only known active and autonomously transposable retroelement in human cells，which is related to autoimmune diseases and plays important roles in activating and regulating the antiviral innate immunity of cells，especially the level of interferon（IFN）. This paper reviews the mechanisms of several non ⁃ structural proteins from human immunodeficiency virus（HIV），hepatitis B virus（HBV）and other viruses participating in the regulation of LINE ⁃ 1 activity. These mechanisms not only ensure the normal expression of viral genome，but also participate in the cellular innate immunity regulation，the inhibition of which may provide new strategies to develop treatments of diseases caused by viruses.

Investigation of Hepatitis B Virus X Gene Mutations in Patients with and without Cirrhosis/Hepatocellular Carcinoma

Background: Chronic HBV (CH) infection and its consequences including cirrhosis (C) and Hepatocellular Carcinoma (HCC) still represent a major Global health. The relationship between HCC and various mutations of HBx gene has been reported. In the present study, we aimed to determine the sequence variation of HBx gene in patients with Chronic HBV infection or C/HCC. Materials and Methods : In this cross-sectional study, 15 patients with HBV chronic infection and 13 with C/HCC were included. After viral DNA extraction using commercial kit HBX gene was amplified using an in-house nestedPCR. Then, bi-directional sequencing was performed on the PCR product. The data resulting from sequencing were aligned with reference HBV sequence to identify the mutations. Results : The mean age of CH and C/HCC groups was 38.23�.46 and 50.67�.22 years old, respectively. We found 43 and 20 Amino acid substitutions inside the region of 88�4 from HBx protein in CH and C/HCC groups, respectively. In addition, K130M+V131I mutation was found in 13.34% (2/15) and 30.7% (4/13) of patients in the CH and C/HCC groups, respectively (P=0.36). Furthermore, 10 deletion mutations were observed in both groups with no significant difference (P=0.8). Conclusion : The results of the present study indicated the relatively high frequency of Amino acid substitutions and deletion, especially in part of region 88-154 from HBx Protein in patients with CH and C/HCC. The findings should be considered in a larger population

Cordyceps sinensis extracts attenuates HBx-induced mesangial cell proliferation and extracellular matrix synthesis by suppression of the PI3K/Akt pathways / 中国临床药理学与治疗学

AlM: To investigate the effect of C. sinensis extracts on HBx (Hepatitis B virus X protein) induced cell proliferation and extracellular matrix (ECM) accumulation and the underlying mechanism in cultured human mesangial cells (HMCs). METHODS: Human mesangial cells were stable transfected with pCMV-HBx to establish an HBx over-expression model, and a control group was transfected with an empty vector. Cell proliferation was determined by MTT assay and DNA synthesis assay. Western blotting was used to measure the expression of PI3K/Akt signaling pathway-related proteins and extracellular matrix. RESULTS: HBx transfection induced cell proliferation, matrix accumulation. HBx-transfected mesangial cells had increased activity of the PI3K/Akt pathways, and treatment with C. sinensis suppressed this effect. CONCLUSlON: C.sinensis attenuates the HBx-induced human mesangial cell proliferation and matrix production in human mesangial cells via inhibition of the PI3K/Akt pathways.

Mechanism of HBx Protein Inhibiting DKK4 Expression and Its Effect on Proliferation and Migration of Hepatocarcinoma Cell Lines / 肿瘤防治研究

Objective To explore the mechanism of hepatitis B virus X protein down-regulating DKK4 and its effect on the proliferation, migration of HCC cell lines. Methods HCC cell lines HepG2 and SMMC7721 cells were infected with adenovirus encoding hepatitis B virus X protein (Ad-HBx), and GFP adenovirus (Ad-GFP) was designed as a control group. We used deacetylase inhibitor (TSA) to treat HCC cell lines and transfected HCC cell lines with small interfering RNA-histone deacetylase 1 (si-HDAC1) and lentivirus overexpressing DKK4. Western blot was used to detect the expression of DKK4, HDAC1 and SIRT1. The proliferation and migration ability of HCC lines were assessed using MTT, crystal violet experiment and Transwell experiment. Results DKK4 expression level was significantly downregulated after Ad-HBx infection (P < 0.05), and its expression level was recovered after TSA treatment (P < 0.05). After silencing HDAC1 with small interfering RNA, the expression of DKK4 could be restored (P < 0.05), the proliferation and migration of HDAC1-silencing or/and DKK4-overexpressing cells decreased (P < 0.05). Conclusion Hepatitis B virus X protein inhibits the expression of DKK4 protein by up-regulating HDAC1 and SIRT1. Silencing HDAC1 and over expressing DKK4 protein could inhibit the proliferation and migration of HCC cell lines infected with Ad-HBx.

Study on the effect and mechanism of hepatitis B virus X protein transactivates gene 4 in HepG2 cell apoptosis / 中华肝脏病杂志

Objective@#To investigate the effect and mechanism of XTP4 gene in apoptotic hepatoblastoma HepG2 cell line.@*Methods@#HepG2 cells were transiently transfected with small interfering RNA of XTP4 genes, plasmid pcDNA3.1/myc-His(-) A-XTP4, and hepatitis B virus X protein transactivated x gene 4 (HBX protein trans-activate gene4, XTP4) and their respective negative controls. After 48h, the overexpression and interference expression condition of XTP4 in HepG2 cells were detected by Western blot. HepG2 cells apoptosis was detected by flow cytometry. The expression levels of apoptosis-related proteins P53, Bcl-2, Bax and Caspase-3 in HepG2 cells were detected by Western blot, and Bcl-2/Bax ratio was calculated. The chemiluminescence assay was used to detect activity of caspase-3 in HepG2 cells. The measured data were presented as ( ± s), and independent sample t-test was used for comparison between the two groups.@*Results@#HepG2 cells had successfully achieved the overexpression and interference expression of XTP4 protein. Compared with the control group, the overexpression of XTP4 in HepG2 cells had significantly decreased the number of apoptotic cells (P < 0.05), and increased Bcl-2/Bax (P < 0.05) ratio, but decreased the expression of P53 protein (P < 0.05). The protein expression of Caspase-3 and activity of caspase-3 was decreased (P < 0.05). However, interference with XTP4 expression in HepG2 cells had significantly increased the number of apoptotic cells (P < 0.05) and decreased Bcl-2/Bax (P < 0.05) ratio, but increased the expression of P53 protein (P < 0.05). The protein expression of Caspase-3 and activity of caspase-3 was increased (P<0.05).@*Conclusion@#In HepG2 apoptosis XTP4 has inhibitory effect, and its effect on inhibiting HepG2 apoptosis may be achieved by regulating the Bcl-2/Bax ratio, and the P53 protein may be involved.

Antiproliferative effect of urolithin A, the ellagic acid-derived colonic metabolite, on hepatocellular carcinoma HepG2. 2. 15 cells by targeting Lin28a/let-7a axis

An abnormality in the Lin28/let-7a axis is relevant to the progression of hepatitis B virus (HBV)-positive hepatocellular carcinoma (HCC), which could be a novel therapeutic target for this malignant tumor. The present study aimed to investigate the antiproliferative and anti-invasive effects of urolithin A in a stable full-length HBV gene integrated cell line HepG2.2.15 using CCK-8 and transwell assays. The RNA and protein expressions of targets were assessed by quantitative PCR and western blot, respectively. Results revealed that urolithin A induced cytotoxicity in HepG2.2.15 cells, which was accompanied by the cleavage of caspase-3 protein and down-regulation of Bcl-2/Bax ratio. Moreover, urolithin A suppressed the protein expressions of Sp-1, Lin28a, and Zcchc11, and elevated the expression of microRNA let-7a. Importantly, urolithin A also regulated the Lin28a/let-7a axis in transient HBx-transfected HCC HepG2 cells. Furthermore, urolithin A decelerated the HepG2.2.15 cell invasion, which was involved in suppressing the let-7a downstream factors HMGA2 and K-ras. These findings indicated that urolithin A exerted the antiproliferative effect by regulating the Lin28a/let-7a axis and may be a potential supplement for HBV-infected HCC therapy.

Kaempferol inhibits cell proliferation, invasion and migration via Wnt/β-catenin signaling in HBx-HepG2 cells / 中国病理生理杂志

AIM: To explore the effects of kaempferol on the proliferation, invasion and migration abilities of HBx-HepG2 cells and to examine the underlying molecular mechanisms.METHODS: The expression levels of related genes at mRNA and protein levels were determined by RT-qPCR and Western blot.The cell apoptotic rate was analyzed by flow cytometry.The cell proliferation, growth, invasion and migration abilities were measured by MTT assay, colony formation assay, Transwell invasion assay and wound healing assay, respectively.RESULTS: Kaemferol inhibited HBx-HepG2 cell proliferation in a concentration-and time-dependent manner.Kaempferol at 100 μmol/L significantly inhibited the colony formation, invasion and migration abilities of the HBx-HepG2 cells.Kaemferol at 100 μmol/L also increased cell apoptotic rate, increased the protein levels of cleaved caspase-3, cleaved caspase-9 and Bax, and decreased the expression level of Bcl-2.In addition, kaemferol at 100 μmol/L suppressed the mRNA and protein expression levels of β-catenin, c-Myc and cyclin D1 in the HBx-HepG2 cells.Kaemferol at 100 μmol/L also suppressed the protein level of p-GSK-3β and the β-catenin protein levels in both cytoplasm and nucleus.LiCl treatment reversed the inhibitory effect of kaempferol on the growth, invasion and migration of the HBx-HepG2 cells.CONCLUSION: Kaempferol inhibits cell proliferation, invasion and migration via activating Wnt/β-catenin signaling in HBx-HepG2 cells.

Expression and significance of HBx protein in HBV-related hepatocellular carcinoma and its impacts on prognosis / 中华普通外科杂志

Objective To explore the relationship between the expression of HBx protein in HBV-related HCC samples and the clinical implications.Methods Elivision two-step was used in this study to detect the expression level of HBx protein in 40 HCC tissues,corresponding para-tumorous tissues from patients with HBV-related HCC undergoing curative hepatectomy.The relationship between HBx protein and clinical parameters (such as gender,age,TNM stage,HBV-DNA load,AFP,liver cirrhosis,a merger of vascular invasion,tumor infiltrating lymphocytes,Edmondson-Steiner histopathological grading,with or without relapse within 24 months) were analyzed.Results (1) The expression of HBx protein in the tumorous tissues was significantly lower than that of para-tumorous tissues (P ＜ 0.05).(2) In the tissues of para-tumorous,the expression of HBx protein in group HBV-DNA ＜ 500 IU/ml was significantly lower than that of group HBV-DNA≥500 IU/ml (P ＜0.05).There were no significant differences in the expression of HBx protein irrespective of gender,age,cirrhosis and the AFP level.(3) In the tissues of tumorous,the expression of HBx protein in group with vascular invasion was significantly higher than that of group without vascular invasion (P ＜ 0.05).However,there were no significant differences in the expression of HBx protein among the factors of TNM stage and Edmondson-Steiner histopathology grading.(4) In para-tumorous tissues,the expression of HBx protein in group of lymphocytic infiltration was significantly higher than that without lymphocytic infiltration (P ＜ 0.05).In the tissues of tumorous,the expression of HBx protein in disease-free survival (DFS) ＜ 24M patients was significantly higher than DFS ≥ 24M (P ＜ 0.05).Conclusions High HBx expression in tumor tissues indicates poor prognosis while that in para-tumorous tissues predicts a better prognosis.

Hepatitis B virus inhibits expression of liver cell immune checkpoint PD-1 ligand gene / 重庆医学

Objective To analyze the impact of hepatitis B virus (HBV) replication and its coded X gene (HBx) expression on cell gene expression profile,especially the immune related gene expression level changes.Methods Overexpressed HBx gene was transiently transfected through lenti-virus and pcDNA3,the RNA-Seq and RT-qPCR methods were used to detect the expression levels of immune related genes,which were verified in HBV replication cell line.Results This study found that the HBV replication and HBx expression suppressed the expression of immune checkpoint PD-1 ligand gene (PD-L1/CD274) in a dose-dependent manner,while the expression of H-box mutant in HBx gene lost this inhibition effect.Conclusion HBV/HBx possesses the ability for inhibiting PD-L1/CD274 ligand gene expression,may relieve the checkpoint of antigen-specific T cell activation in viral infection acute stage,activates cytotoxic T cells,which may cause that T cell attack and clear highly replicated cells,helps virus to enter the lower replication status,and reaches the balance status between virus-host and lays a basis of HBV chronic infection.

Hepatitis B virus inhibits expression of liver cell immune checkpoint PD-1 ligand gene / 重庆医学

Objective To analyze the impact of hepatitis B virus (HBV) replication and its coded X gene (HBx) expression on cell gene expression profile,especially the immune related gene expression level changes.Methods Overexpressed HBx gene was transiently transfected through lenti-virus and pcDNA3,the RNA-Seq and RT-qPCR methods were used to detect the expression levels of immune related genes,which were verified in HBV replication cell line.Results This study found that the HBV replication and HBx expression suppressed the expression of immune checkpoint PD-1 ligand gene (PD-L1/CD274) in a dose-dependent manner,while the expression of H-box mutant in HBx gene lost this inhibition effect.Conclusion HBV/HBx possesses the ability for inhibiting PD-L1/CD274 ligand gene expression,may relieve the checkpoint of antigen-specific T cell activation in viral infection acute stage,activates cytotoxic T cells,which may cause that T cell attack and clear highly replicated cells,helps virus to enter the lower replication status,and reaches the balance status between virus-host and lays a basis of HBV chronic infection.

miR-222 enhances HBx-HepG2 cell growth via regulation of BCL2L13 gene / 中国病理生理杂志

AIM:To investigate the regulation of miR-222 on BCL2L13 gene and its effect on the growth and apoptosis of HBx-HepG2 cells, and to explore the underlying molecular mechanisms .METHODS:The expression level of miR-222 was detected by RT-qPCR.The HBx-HepG2 cell growth was examined by MTT and colony formation assays .The cell cycle and apoptosis were analyzed by flow cytometry .The recombination vector pmirGLO-BCL2L13 was constructed, and dual-luciferase reporter experiment was performed to validate the target of miR-222.RESULTS:The expression level of miR-222 in the HBx-HepG2 cells was significantly higher than that in the L 02 cells ( P<0.05 ) .Over-expression of miR-222 enhanced HBx-HepG2 cell growth, changed cell cycle, and inhibited apoptosis (P<0.05).Knockdown of miR-222 reduced HBx-HepG2 cell growth, changed cell cycle, and increased cell apoptotic rate (P<0.05).BCL2L13 was down-regulated in the HBx-HepG2 cells as compared with L02 cells (P<0.05), and knockdown of miR-222 in the HBx-HepG2 cells increased the expression level of BCL2L13 (P<0.05).The results of dual-luciferase reporter assay and re-store experiment showed that miR-222 negatively regulated the expression of BCL2L13 via targeting 3’ UTR of BCL2L13, resulting in the promotion of HBx-HepG2 cell growth .CONCLUSION: miR-222 enhances HBx-HepG2 cell growth via down-regulation of BCL2L13.

Synthesis of 8-bromo-ethoxy Rhein and evaluation of its inhibition effect on hepatitis B virus in human hepatoma cells HepG2.2.15 / 中国药理学通报

Aim To synthesize 8-bromo-ethoxy Rhein and investigate its mechanisms and inhibition effect on hepatitis B surface antigen ( HBsAg ) and e antigen ( HBeAg) in HepG2.2.15 cells.Methods 8-bromo-ethoxy Rhein was synthesized based on the chemical structure of Rhein , and its structure was identified by IR,1 H-NMR and 13 C-NMR spectra.MTT assay was used to test the inhibitory effect of 8-bromo-ethoxy Rhein on HepG2.2.15 cells.After the cells treatment by 8-bromo-ethoxy Rhein , the HBsAg and HBeAg in cell supernatant were detected by ELISA .The expres-sion of hepatitis B virus X gene ( HBx) was detected by Western blot .The cell cycles were examined with flow cytometry.The intracellular free calcium concentration was detected by laser scanning confocal microscopy . Results The structure of 8-bromo-ethoxy Rhein was confirmed by IR,1 H-NMR and 13 C-NMR.MTT results showed that synthetic product and Rhein could inhibit the cell proliferation in HepG2.2.15 cells.After trea-ted with 8-bromo-ethoxy Rhein and Rhein for 72 h,the half inhibitory concentration 50%( IC50 ) was 14.29 mg? L-1 and 11.59 mg? L-1 , respectively .Using non-cytotoxic dose of 8-bromo-ethoxy Rhein , the inhibitory effect on HBsAg and HBeAg was gradually enhanced with increasing 8-bromo-ethoxy Rhein concentration . The inhibitory effect of synthetic product on hepatitis B virus was better than that of Rhein .8-bromo-ethoxy Rhein could down-regulate the expression of HBx , in-tracellular calcium ion concentration and block the hepatitis B virus ( HBV ) replication.Flow cytometry results showed 8-bromo-ethoxy Rhein didn′t affect the cell cycle .Conclusions Compare with Rhein , the synthesis of 8-bromo-ethoxy Rhein shows stronger inhi-bition on hepatitis B virus in HepG2.2.15, and its mechanisms may involve down-regulating the expres-sion of HBx and reducing calcium ion concentration .

Hepatitis B virus X protein induces epithelial-mesenchymal transition through c-Src activation in liver cancer cells / 中华肝胆外科杂志

Objective To examine the role of c-Src activation in hepatitis B virus X (HBx) protein induced epithelial-mesenchymal transition (EMT) in liver cancer.Methods SMMC-7721 liver cancer cells were transfected with HBx gene to induce EMT and the activated c-Src expression was evaluated by Western blot.Both the morphological changes and the epithelial and mesenchymal markers expression (real-time PCR,western blot and immunocytochemistry) of HBx-transfected SMMC-7721 cell treated by c-Src kinase inhibitor PP2 and negative control PP3 were observed and compared,respectively.Results The activated c-Src expression in HBx gene transfected SMMC-7721 cells was significantly increased compared to that in mock transfected cells,c-Src kinase inhibitor PP2 could enable the HBx-transfected SMMC-7721 cells to transmit from spindle-like shape to original epithelial morphology.Western blot and immunocytochemistry confirmed that the expression of epithelial markers and mesenchymal markers almost returned to the levels of parental cells,indicating the mesenchymal-epithelial transition.Conclusions c-Src activation plays a key role in the process of EMT induced by HBx protein in SMMC-7721 cells.

The Effect of Metformin Treatment on CRBP-I Level and Cancer Development in the Liver of HBx Transgenic Mice

Retinoids regulate not only various cell functions including proliferation and differentiation but also glucose and lipid metabolism. After we observed a marked up-regulation of cellular retinol-binding protein-I (CRBP-I) in the liver of hepatitis B virus x antigen (HBx)-transgenic (HBx Tg) mice which are prone to hepatocellular carcinoma (HCC) and fatty liver, we aimed to evaluate retinoid pathway, including genes for the retinoid physiology, CRBP-I protein expression, and retinoid levels, in the liver of HBx Tg mice. We also assessed the effect of chronic metformin treatment on HCC development in the mice. Many genes involved in hepatic retinoid physiology, including CRBP-I, were altered and the tissue levels of retinol and all-trans retinoic acid (ATRA) were elevated in the liver of HBx Tg mice compared to those of wild type (WT) control mice. CRBP-I protein expression in liver, but not in white adipose tissue, of HBx Tg mice was significantly elevated compared to WT control mice while CRBP-I protein expressions in the liver and WAT of high-fat fed obese and db/db mice were comparable to WT control mice. Chronic treatment of HBx Tg mice with metformin did not affect the incidence of HCC, but slightly increased hepatic CRBP-I level. In conclusion, hepatic CRBP-I level was markedly up-regulated in HCC-prone HBx Tg mice and neither hepatic CRBP-I nor the development of HCC was suppressed by metformin treatment.

Establishment of human HBV/HBx-Knockin-p53 rat embryonic stem cell line / 第二军医大学学报

Objective To establish human HBV/HBx-knockin-p53 rat embryonic stem (ES) cell line, so as to lay a foundation for establishment of animal models. Methods First we constructed the targeting vector pKO-gHBV or pKO-X with HBV whole genome or X and p53 homologue arms by PCR and connection with common gene targeted vector pKO; then after linearized with Sal I the vector was transferred into rat ES cells through electroporation. And 2i ES culture medium containing puromycin was used for a three-cycle selection of puromycin resistant clones. Then PCR was used to screen the obtained positive clones; mycoplasma examination and karyotypic analysis were used to confirm targeted ES cell clones. Results We successfully obtained two targeted vectors: pKO-gHBV and pKO-X; then after three-cycle drug-selection we obtained 2 pKO-X clones and 1 pKO-gHBV clone, which were confirmed by PCR, mycoplasma examination and karyotypic analysis. Conclusion We have successfully established human HBV/HBx-knockin-p53 rat ES cells, paving a way for future establishment of HBV/HBx-knockin-p53 rat model.

Could HBx Protein Expression Affect Signal Pathway Inhibition by Gefitinib or Selumetinib, a MEK Inhibitor, in Hepatocellular Carcinoma Cell Lines?

Hepatitis B virus X (HBx) protein has been known to play an important role in development of hepatocellular carcinoma (HCC). The aim of this study is to find out whether HBx protein expression affects antiproliferative effect of an epidermal growth factor receptor-tyrosine kinase (EGFR-TK) inhibitor and a MEK inhibitor in HepG2 and Huh-7 cell lines. We established HepG2 and Huh-7 cells transfected stably with HBx gene. HBx protein expression increased pERK and pAkt expression as well as beta-catenin activity in both cells. Gefitinib (EGFR-TK inhibitor) inhibited pERK and pAkt expression and beta-catenin activity in both cells. Selumetinib (MEK inhibitor) reduced pERK level and beta-catenin activity but pAkt expression was rather elevated by selumetinib in these cells. Reduction of pERK levels was much stronger with selumetinib than gefitinib in both cells. The antiproliferative efficacy of selumetinib was more potent than that of gefitinib. However, the antiproliferative effect of gefitinib, as well as selumetinib, was not different between cell lines with or without HBx expression. Signal pathway activation by HBx might not be strong enough to attenuate the antiproliferative effect of EGFR-TK inhibitor. Future experiments are needed to understand the role of HBx protein expression in HCC treatment using molecular targeting agent.

Effects of hepatitis B virus X protein on cell proliferation and GSK3β expression of human liver cell line L02 / 肿瘤

Objective: To investigate the effects of hepatitis B virus (HBV) X protein (HBx) on cell proliferation, cell cycle and the glycogen synthase kinase 3β (GSK3β) expression of human liver cell line LO2, and discuss the mechanism of carcinogenesis of HBV-associated hepatocellular carcinoma (HCC). Methods: The human liver cell line L02 was infected with Ad-GFP or Ad-HBx. The expressions of HBx and GSK3β mRNAs were identified by RT-PCR. MTT method and flow cytometry were used to detect the cell proliferation and cell cycle distribution, respectively. The expression levels of HBx, total-GSK3β (t-GSK3β),phospho-GSK3β (p-GSK3β), β-catenin and cyclin D1 proteins were examined by Western blotting. Results: The positive expressions of HBX mRNA and protein in L02 cells were detected after infection with Ad-HBx. The cell proliferation rate was elevated in a time-dependant manner. The percentage of Ad-HBx-infected cells at G1 period was lower than that in the control group, while the percentages of Ad-HBx-infected cells at S and G2 periods were both higher than those in the control group (P<0.05). The expression levels of t-GSK3β mRNA and protein in Ad-HBx-infected cells had no significant changes, and the expression levels of p-GSK3β, β-catenin and cyclin D1 proteins were increased compared with those in the control group (P<0.05). Conclusion: HBx may participate in the activation of downstream Wnt/β-catenin signal pathway through promoting the phosphorylation of GSK3β in L02 cells, and eventually increase the cell proliferation ability.

Screening and identification of HLA-A0201 restricted CTL epitope from hepatitis B virus X protein in vitro / 第二军医大学学报

Objective: To identify the HLA-A0201 restricted CTL epitopes derived from hepatitis B virus X protein predicted by computer program and general principles in vitro. Methods: HBx gene sequences of Hepatitis B virus genotypes B/C and serotypes adw/adr, with the highest frequencies in Chinese, were computed and analyzed by screening service offered by Internet combined with peptide supermotif, extended motif and quantitative motif prediction. Four most ideal nine-peptides (HBx1, HBx2, HB3x3, and HBx4) were selected as candidate peptides. Using flow cytometry, the fluorescence index of both control and experimental groups were detected and the 4 nine-peptides were evaluated with T2 binding assay and DC50 assay. Results: The nine-peptides VLCLRPVGA (HBx1), CLFKDWEEL (HBx2), VLHKRTLGL (HBx3) and HLSLRGLPV (HBx4) were selected as candidate targets. Among the 4 candidate peptides, HBx2 showed higher HLA-A0201 affinity and HBx2, HBx4 showed better stability. Conclusion: Our study indicates that CLFKDWEEL might be a potential HLA-A0201 restricted CTL epitope from hepatitis B virus X protein; further study is needed for verification of its immunity in vivo.