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.

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 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.

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.

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.

Effect of hepatitis B virus X protein on nuclear localization and transcription of ?-catenin in human liver cell / 基础医学与临床

Objective To investigate the interaction between HBx protein and ?-catenin for exploring molecular mechanism in HBV-associated hepatocellular carcinoma.Methods Iimmunocytochemistry and Luciferase Assay were used to detect the endocellular location and transcriptional activity of ?-catenin in normal liver cell line L02 infected by Ad-HBx.Results Immunocytochemistry: there was markedly increased expression of ?-catenin in cytoplasm and nucleus.Luciferase Assay: Ad-HBx led to a significant increase of TCF-4-dependent transcription,the TOP count of Ad-GFP and Ad-HBx-GFP was 1.4 fold and 11 fold as compared to the negative control respectively(P