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ObjectiveTo investigate whether cytotoxic T lymphocyte （CTL）－derived exosomes can downregulate HBx expression and inhibit hepatic stellate cell （HSC） activation. MethodsThe supernatants of HepG2， HepGA14， and CTL cells were collected to extract exosomes， which were referred to as NC－exo， HBV－exo， and CTL－exo， respectively）. Transmission electron microscopy was used to observe their morphology， and Western Blot was used to measure the expression of the markers of exosomes CD63 and TSG101. NC－exo， HBV－exo， and CTL－exo labeled by BODIPY dye were mixed with HBV－exo at different ratios and were then co－cultured with HSC LX－2 （HSC－LX2）. A fluorescence microscope was used to observe whether exosomes could enter LX－2 cells， and an fluorescence microscope was used to observe cell morphological changes； quantitative real－time PCR （qPCR） was used to measure the expression of the activated biomarkers such as transforming growth factor－β1 （TGF－β1）， ɑ－smooth muscle actin （ɑ－SMA）， and collagen type I （Collagen I） in LX－2 cells. CTL－exo was added to the HepGA14 culture system； then qPCR was used to measure the mRNA expression level of HBV DNA， cccDNA， and HBx in exosomes in HepGA14 cells， and Western Blot was used to measure the protein expression level of HBx in exosomes. The t-test was used for comparison of normally distributed continuous data between two groups； a one-way analysis of variance was used for comparison between multiple groups， and the least significant difference t-test was used for further comparison between two groups. ResultsThe exosomes were all microcysts with a double－layer membrane structure and were circular or elliptical in shape， with the expression of the signature proteins CD63 and TSG101， and the vesicles had a diameter of 50－100 nm. The fluorescence microscope showed that exosomes could enter LX－2 cells， and HSC were enlarged with extended cell processes. The results of qPCR showed that there were significant differences in the expression levels of TGF－β1， ɑ－SMA， and Collagen I genes between the NC－exo， HBV－exo， NC－exo+HBV－exo， and Con groups （F=444.678， 417.144， and 571.508， all P<0.05）. After the intervention of HepGA14 cells with CTL－exo， qPCR results showed that compared with the control group， there were significant reductions in the expression levels of HBV DNA and cccDNA in HepGA14 cells （all P<0.05）， the relative mRNA expression level of HBx in exosomes （P<0.05）， and the protein expression level of HBx （P<0.05）. CTL－exo and HBV－exo were mixed at different ratios （2∶1， 5∶1， 10∶1） and were then used for the intervention of LX－2 cells， and qPCR results showed that the expression levels of TGF－β1， ɑ－SMA， and Collagen I genes in LX－2 cells gradually decreased with the increase in the ratio of CTL－exo between groups （P<0.05）. ConclusionCTL－exo can downregulate the protein expression of HBx in HBV－exo to inhibit HSC activation， suggesting that CTL－exo has an anti－hepatitis B liver fibrosis effect.
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Objective:To investigate the effects of the interaction between ubiquitin-specific peptidase 22 (USP22) and hepatitis B virus X protein (HBx) on the protein level and the biological function of HBx.Methods:The interactions between HBx and USP22 were analyzed by GST pull-down, co-immunoprecipitation assay and confocal laser scanning assay. USP22 recombinant plasmids or specific siRNA were transiently co-transfected with HBx plasmids. Western blot were used to detect the protein level of HBx. The half-life and degradation pathway of HBx in the transfected cells treated with cycloheximide (CHX) or proteasome inhibitor MG132 were detected. In vivo ubiquitination assay was used to detect the ubiquitination of HBx with USP22 overexpression. Moreover, dual-luciferase reporter assay and colony formation assay were used to analyze the effects of USP22 on the biological function of HBx. Results:USP22 could interact with HBx in vivo and in vitro. USP22 significantly increased the stability of HBx and inhibited the proteasome-mediated degradation of HBx protein by reducing the ubiquitination of HBx, thereby enhancing the biological function of HBx. Conclusions:USP22 inhibited HBx protein degradation through ubiquitin-dependent proteasome pathway, thus enhancing the stability and biological function of HBx.
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Objective:To investigate whether hepatitis B virus X protein (HBx) mediates the podocyte injury through reactive oxygen species (ROS) /nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) signaling pathway.Methods:HBx-overexpressing lentivirus was transfected into renal podocytes of mouse to mimic the pathogenesis of hepatitis B virus-associated glomerulonephritis. Podocytes were divided into the following five groups: blank control group (no special treatment), negative control group (transfected with control lentivirus), HBx overexpression group (transfected with HBx overexpression lentivirus), HBx overexpression+NLRP3 siRNA group (transfected with HBx overexpression lentivirus and NLRP3 siRNA), and HBx overexpression+ROS inhibitor group (transfected with HBx overexpression lentivirus and adding ROS inhibitor). The morphological changes of podocytes were observed with electron microscope. The generation of ROS was detected by dichlorodihydrofluorescein diacetate assay (DCFH-DA). Hoechst 33342 staining was used to observe the morphological and quantitative changes of podocyte nuclei. Enzyme-linked immunosorbent assay was used to detect caspase-1 activity, and the levels of lactate dehydrogenase, interleukin (IL)-1β and IL-18. Quantitative real-time polymerase chain reaction and Western blotting were used to detect the expression levels of mRNA and protein of pyroptosis-related protein, such as NLRP3, apoptosis-associated speck-like protein containing card (ASC), caspase-1, IL-1β and IL-18. TUNEL staining and flow cytometer were used to detect the number of pyroptosis cells. Immunofluorescence staining was used to detect the expression levels of desmin and nephrin.Results:After successful infection of podocytes with HBx-overexpressing lentivirus, pyroptosis-related morphological changes in the cells were observed under electron microscope. The level of ROS in the HBx overexpression group was significantly higher compared to the negative control group ( P<0.05). Hoechst 33342 staining revealed condensed nuclei in the HBx overexpression group. TUNEL staining and flow cytometer demonstrated that podocytes underwent increased pyroptosis in the HBx overexpression group. The mRNA and protein expression levels of pyroptosis-related proteins such as NLRP3, ASC, caspase-1, IL-1β and IL-18 were up-regulated upon HBx overexpression (all P<0.05). Caspase-1 enzyme activity, lactate dehydrogenase and desmin expression levels were enhanced after HBx overexpression (all P<0.05). However, NLRP3 knockdown or addition of ROS inhibitors attenuated the pyroptosis and increased expression levels of pyroptosis-related proteins caused by HBx overexpression (all P<0.05). Conclusion:ROS/NLRP3 pathway plays an important role in HBx-induced podocyte pyroptosis.
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Objective:To investigate the effects of hepatitis B virus X (HBx) on hepatocellular carcinoma (HCC) proliferation, invasion, and sorafenib resistance.Methods:HepG2 cell line infected with HBx ORF lentivirus was set as the HBx high expression group and infected with empty vector was set as the negative control group. The interference group was infected with the HBx siRNA virus based on the HBx high expression group to reduce HBx expression. Interference control group as interference group but with infected empty vector virus. Western blotting was used to measure the protein level of HBx. Cell proliferation, invasion ability, and sorafenib semi-inhibitory concentration (IC50) of HCC cells under different HBx expression levels were respectively detected by cell proliferation assay kit, Transwell invasion assay, and cell titer-glo kit.Results:Western blotting showed that the stable cell lines were successfully established. Cell proliferation of the HBx high expression group was better than that of the blank control and negative control groups, and the cell proliferation of the interference group was lower than that of the interference control and HBx high expression groups, and the differences were all statistically significant ( P<0.05). The number of cells crossing Matrigel gel was (46.2±4.1), (50.7±5.1) and (48.2±5.2) in the blank control group, negative control group, and interference group, respectively. The number of cells crossing Matrigel gel in the HBx high expression group (124.2±8.3) and the interference control group (117.2±7.5) were higher than the above three groups, respectively, and the differences were all statistically significant ( P<0.05). The IC50 of cells in the HBx high expression group and the interference control group were (5.36±0.31) μmol/L and (5.48±0.20) μmol/L, respectively, which were higher than those in the blank control group, the negative control group, and the interference group (4.75±0.22) μmol/L, (4.60±0.14) μmol/L and (3.98±0.03) μmol/L. The differences were all statistically significant ( P<0.05). Conclusion:HBx promoted the tumor proliferation and invasion of HepG2 HCC cells, enhanced the ability to sorafenib resistance, and inhibited apoptosis.
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The pathogenesis of hepatitis B virus （HBV）-associated hepatocellular carcinoma （HCC） is complicated with the crosstalk of multiple factors and the multi-step processes. The main mechanisms underlying the HBV-induced HCC include:①integration of HBV DNA into the host hepatocyte genome to alter gene function at the insertion site，resulting in host genome instability and expression of carcinogenic truncated proteins;②HBV gene mutations at S，C，and X coding regions in the genome;③HBV X gene-encoded HBx protein activates proto-oncogenes and inhibits tumor suppressor genes，leading to the HCC occurrence. In this article，the recent research progress on the molecular mechanism of HBV-induced HCC is comprehensively reviewed，so as to provide insights into the prevention，early prediction and postoperative adjuvant therapy of HCC.
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Humans , Carcinoma, Hepatocellular , Hepatitis B/complications , Hepatitis B virus/genetics , Hepatocytes , Liver NeoplasmsABSTRACT
Objective@#To investigate the effects and mechanism of hepatitis B virus x protein (HBx) on human hepatocellular carcinoma cells proliferation.@*Methods@#Eukaryotic expression vector HBx-pEGFP-C1 was constructed. HepG2 cells were transfected transiently using Lipofectamine 2000. HBx expression in transfected cells were measured by RT-PCR and Western blot. Cells proliferation and apoptosis were detected by using growth curves and TUNEL staining. The protein levels of caspase-3, p-p38, p-Akt and p-JNK were measured by Western blot.@*Results@#HBx was successfully expressed in HepG2 cells. Growth curve result showed that HBx promoted cell proliferation (t=-0.8999, P=0.012). Compared with control group, the levels of p-p38(24 h) (t=- 11.058, P=0.0004) and p-JNK(48 h) (t=- 15.022, P=0.0001) in HBx-pEGFP-C1 group were increased significantly. There is no significant difference between the two groups′ apoptosis.@*Conclusions@#Transient overexpression of HBx promoted human hepatic carcinoma cells proliferation and activated the p38 and JNK signalling pathway.
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Objective@#To study the interaction between hepatitis B virus X protein(HBx) and mitochondrial elongation factor G1 (EFG1) in yeast cells or hepatoma cells.@*Methods@#After verification the interaction between HBx and EFG1 by CytoTrap yeast two-hybrid system, EFG1 genome was amplified by means of polymerase chain reaction(PCR) and cloned into the pcDNA3.1/myc-His(-)A vector, following verification by sequencing. Expression of HBx and EFG1 protein was verified in Huh7 cells. Then the recombinant vector pcDNA3.1/myc-His(-)A-EFG1 and pFLAG-CMV-2-HBx were transfected into Huh7 cells; 48 h later, the cells were lysed. The direct interaction between HBx and EFG1 was further confirmed by the Co-immunoprecipitation (Co-IP) assay.@*Results@#The interaction between HBx and EFG1 was successfully verified by CytoTrap yeast two-hybrid system. The recombined plasmid pcDNA3.1/myc-His(-)A-EFG1 was obtained. Furthermore, Co-IP assay was used to confirm the interaction between HBx and EFG1 in Huh7 cells.@*Conclusions@#The direct interaction between HBx and EFG1 was confirmed. Therefore our findings provide experimental basis for the influence of HBx protein on the expression of mitochondrial protein and provide new insights into the pathogenesis of HBx in the development of hepatocellular carcinoma.
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Objective To investigate the effect of occult HBV infection (OBI) on carcinogenesis of cryptogenic hepatocellular carcinoma.Methods Samples of hepatocellular carcinoma (HCC) and pericarcinomatous tissues obtained after hepatectomy from January 2011 to November 2013 at the Third Affiliated Hospital of Sun Yat-Sen University were collected.They were divided into two groups:the cryptogenic HCC group (the CH group,n =26) and the HBV related HCC group (the HH group,n =40).These samples were compared with the normal liver tissues obtained in 30 patients.HBV DNA was identified by the nested polymerase chain reaction and the immunohistochemical method was taken to examine the hepatitis B virus X protein (HBx) and Yes-associated protein (YAP) expressions.Results OBI was identified in 20 (77.8％) cryptogenic HCC patients and 8 (26.7％) in the control group.There was a significant difference between the two groups (x2 =14.072,P ＜ 0.05).HBV DNA was detected in all the HBV-related HCC patients.The HBx protein expression was mainly located in the cytoplasm of liver cells and liver cancer cells,but YAP was expressed in the nucleus.Both of them showed diffuse brown or tan particles.In the HH group and the CH group,the positive expression rates of HBx protein in the tumorous tissues were 80.0％ and 90.0％,respectively,and 85.0％ and 82.5％ in the nontumorous tissues,but only in 40.0％ in the control group.The positive expression rates of YAP in the tumorous tissues were 65.0％ and 67.5％,respectively,15.0％ and 20.0％ in the nontumorous tissues,respectively,but only in 12.5％ in the control group.The HBx expression in the cancerous tissues and para-cancerous tissues of the HH group and the CH group showed no significant difference (P ＞ 0.05),but the YAP expression in the tumor tissues was significantly higher than that in the nontumorous tissues (P ＜ 0.05).The HBx and YAP expressions in the HH group were comparable to the CH group (P ＞ 0.05).However,their expressions in the cancerous tissue of the HH group and the CH group were significantly higher than in the normal liver tissues (P ＜ 0.05).Conclusion A high prevalence of HBV infection was observed in HBsAg-negative HCC and the high expressions of HBx and YAP might be involved in the process of cryptogenic hepatocellular carcinoma.
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Objective To explore the effects and mechanisms of hepatitis B virus-X protein (HBx) on invasion and migration of hepatocellular carcinoma (HCC) cells.Methods The retrospective cohort study was conducted.The clinicopathological data of 30 patients with liver tumor (20 with HCC and 10 with benign tumor of liver) who were admitted to the Affiliated Hospital of Xuzhou Medical College between July 2014 and July 2015 were collected.HCC tissues of 20 patients with HCC (with history of HBV infection) were collected by surgical resection and peritumoral normal tissues (outside of tumor capsule) of 10 patients with benign tumor of liver (without history of HBV infection) were collected.The expressions of epidermal growth factor receptor 3 (ErbB3)in HCC tissues and peritumoral normal tissues were detected by immunohistochemistry (IHC).The relative expressions of ErbB3 and HBx in HCC tissues and peritumoral normal tissues were detected by Western blot,and relative expressions of ErbB3 in HepG2 of which green fluorescent protein (GFP) and GFP-HBx were respectively transfected were detected.The relative expressions of ErbB3 mRNA in HepG2 transfected by GFP and GFP-HBx were detected by real-time polymerase chain reaction (RT-PCR).The migration and invasion of HepG2 were respectively detected by Transwell assay with and without matrix.The measurement data with normal distribution were represented as $± s.The comparisons between groups were evaluated with the independent-sample t test.Correlation analysis was done by the Pearson test.Results (1) The expressions of ErbB3 were detected by IHC:relative value of mean optical density (MOD) of ErbB3 in HCC tissues of 20 patients with HCC and peritumoral normal tissues of 10 patients with benign tumor of liver were 2.54± 1.33 and O.99±0.29,respectively,with a statistically significant difference (t =6.542,P ＜ 0.05).(2) The relative expressions of ErbB3 and HBx were detected by Western blot:relative expressions of ErbB3 and HBx were respectively 0.79±0.13,1.10±0.28 in HCC tissues of 10 patients with HCC and 1.07±0.17,0 in peritumoral normal tissues of 10 patients with benign tumor of liver,with statistically significant differences (t =3.229,19.486,P＜0.05).The results of Pearson test showed that there was a positive correlation of expression between ErbB3 and HBx in HCC tissues (r=O.637,P＜ 0.05).(3) The relative expressions and transcriptional levels of ErbB3 were detected by Western blot and RT-PCR:relative expressions of ErbB3 in HepG2 of which GFP and GFP-HBx were respectively transfected were O.75±0.11 and 1.10±0.10,respectively,with a statistically significant difference (t=4.291,P＜0.05).The relative expressions of ErbB3 mRNA in HepG2 of which GFP and GFP-HBx were respectively transfected were O.38±0.03 and O.94±0.07,respectively,with a statistically significant difference (t=11.703,P＜O.05).(4) The effects of ErbB3 on migration and invasion of HepG2:numbers of transmenbrane cell in HepG2 of which His and His-ErbB3 were respectively transfected by Transwell assay with matrix were respectively 271± 18 and 463± 31,respectively,with a statistically significant difference (t =8.202,P＜0.05).Numbers of transmenbrane cell in HepG2 of which His and His-ErbB3 were respectively transfected by Transwell assay without matrix were respectively 315±38 and 549±34,respectively,with a statistically significant difference (t =8.310,P＜0.05).Conclusion HBx protein can promote the invasion and migration of hepatocellular carcinoma cells through up-regulating expressions of ErbB3 protein.
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BACKGROUND/AIMS: Hepatitis B viral protein X (HBx) is implicated in the pathogenesis of hepatocellular carcinoma (HCC) as well as the elevation of heat shock proteins (HSPs) after hepatitis B virus (HBV) infection. We thus investigated the anticancer effects of an HSP90 inhibitor 17-Dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) in HBx-transfected hepatocellular carcinoma cells. METHODS: pcDNA-HBx was made by inserting the HBx gene derived from the HBV-infected patient into pcDNA3.1 using the restriction enzymes (XbaI/HindIII). HBx-expressing HepG2 cells were then generated by transfecting HepG2 cells with pcDNA containing HBx gene. To compare the anticancer effects of 17-DMAG between pcDNA-HBx transfected HepG2 cells and the control cells (pcDNA-transfected HepG2 cells), we performed various molecular studies, including Ez-cytox proliferation assay, Western blot analysis, and flow cytometry. RESULTS: 17-DMAG inhibited the proliferation of pcDNA-HBx transfected HepG2 cells better than control cells (P<0.05). After treating with a various concentration of 17-DMAG (50–1,000 nM), pcDNA-HBx transfected HepG2 cells exhibited higher expression of pro-apoptotic proteins (c-caspase-3, c-caspase-8, and c-caspase-9) than did control cells (P<0.05). pcDNA-HBx transfected HepG2 cells showed higher activities of caspase-3, caspase-8, and caspase-9 than did control cells (P<0.05). Finally, we found that the expression of pro-apoptotic proteins (PARP and c-caspase-3) was considerably decreased by the use of a caspase inhibitor suggesting that 17-DMAG induces the cell death of HepG2 cells caspase-dependently. CONCLUSIONS: Our study strongly suggests that 17-DMAG has antiviral effects against HBV as well as anticancer effects against HepG2 cells. Thus, the application of 17-DMAG appears to be particularly advantageous to the HCC patients related with HBV infection.
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Humans , Apoptosis , Apoptosis Regulatory Proteins , Blotting, Western , Carcinoma, Hepatocellular , Caspase 3 , Caspase 8 , Caspase 9 , Caspases , Cell Death , Flow Cytometry , Heat-Shock Proteins , Hep G2 Cells , Hepatitis B virus , Hepatitis B , Hepatitis , TransfectionABSTRACT
Liver cancer occurs very frequently worldwide and hepatocellular carcinoma (HCC) accounts for more than 80% of total primary liver cancer cases. In this study, the anticarcinogenic effects of resveratrol against hepatitis B virus (HBV)-induced HCC were investigated by using HBV X-protein-overexpressing Huh7 (Huh7-HBx) human hepatoma cells. MTT assay showed that resveratrol decreased cell viability. Fluorescence-activated cell-sorter analysis showed that resveratrol induced G1 cell cycle arrest without increasing the sub-G1 phase cell population. Therefore, we evaluated its effect on regulation of cyclin D1, which is critically involved in G1/S transition. Resveratrol lowered cyclin D1 transcription. Western blot analysis of the effects of resveratrol on upstream cyclin D1 transcriptional signaling, extracellular signal-related kinase (ERK), p90(RSK), Akt, and p70(S6K) revealed inhibition of Akt but not the ERK signaling pathway. Collectively, the results indicate that resveratrol inhibits Huh7-HBx proliferation by decreasing cyclin D1 expression through blockade of Akt signaling. We investigated the anticarcinogenic effect and mechanism of resveratrol in xenograft model mice implanted with Huh7-HBx cells. Intraperitoneal resveratrol injection reduced tumor size in the mice. Expression of survivin was reduced, but cyclin D1 was not affected. The results demonstrate that resveratrol treatment may help manage HBV-induced HCC by regulating survivin.
Subject(s)
Animals , Humans , Mice , Anticarcinogenic Agents , Blotting, Western , Carcinoma, Hepatocellular , Cell Survival , Cyclin D1 , G1 Phase Cell Cycle Checkpoints , Hepatitis B virus , Hepatitis B , Hepatitis , Heterografts , Liver Neoplasms , Phosphotransferases , Ribosomal Protein S6 Kinases, 90-kDaABSTRACT
Objective To investigate the influence of hepatitis B virus X (HBx) protein on the activity of NLRP3 inflammasome as well as the mechanism of accelerating HBV-related hepatocellular carcinoma (HCC).Methods The HepG2 cell strains were divided into the 5 groups:blank control group (without plasmid transfection),empty vector group [transfected with pE green fluorescent protein (GFP)-N1 vector plasmid],fulllength HBx protein group (transfected with pEGFP-N1-X plasmid),HBx1-127 group (transfected with pEGFP-N1-X1-127 plasmid),HBx1-101 group (transfected with pEGFP-N1-X1-101 plasmid).(1) The expressions of HBx protein and NLRP3 inflammasome were detected by Western blot [Lipopolysaccharide (LPS) + ATP intervention was performed in the blank control group].(2) The HepG2 cells in the full-length HBx protein group were respectively intervened by glibenclamide and ammonium pyrrolidinedithiocarbamate (APDC),and the expressions of IL-1β and IL-18 were detected by enzyme-linked immunosorbent assay (ELISA).(3) The expressions of reactive oxygen were detected by flow cytometry.The measurement data with normal distribution were presented by (x) ± s.The one-way ANOVA was adopted in the comparison among groups while the t test was used in the pairwise comparison.Results (1) The results of Western blot showed:① the relative expressions of HBx recombinant plasmid fusion protein inside the HepG2 cells in the blank control group,empty vector group,full-length HBx protein group,HBx1-127 group and HBx1-101 group were 0.07 ±0.03,0.92 ±0.13,0.84 ±0.11,0.30 ±0.06 and 0.29 ± 0.05,respectively.The expressions in the HBx1-127 group and the HBx1-101 group represented the expressions of HBx1-127 protein and HBx1-101 protein.There were statistically significant differences among the 5 groups (F =61.790,P ＜ 0.05).The relative expression of full-length HBx protein group was significantly different from that of blank control group,HBx1-127 group and HBx1-101 group (t =12.070,7.465,7.801,P ＜0.05).There was no statistically significant difference between full-length HBx protein group and empty vector group (t =0.867,P ＞0.05) and between the HBx1-127group and HBx1-101 group (t =0.146,P＞0.05).② The relative expression of NLRP3 inflammasome protein inside the HepG2 cells in the blank control group,full-length HBx protein group,HBx1-127 group,HBx1-101 group and LPS + ATP group were 0.29 ±0.06,0.83 ±0.14,0.27 ±0.06,0.27 ± 0.05 and 0.90 ± 0.16,respectively,with a statistically significant difference among the 5 groups (F =29.550,P ＜ 0.05).The relative expression of NLRP3 inflammasome protein of LPS + ATP group was significantly different from that of blank control group,HBx1-127 group and HBx1-101 group,respectively (t =6.310,6.565,6.741,P ＜0.05).There were statistically significant differences between the full-length HBx group and the HBx1-127 group or HBx1-101 group (t =6.381,6.584,P ＜ 0.05) and no statistically significant difference between LPS + ATP group and full-length HBx protein group (t =0.580,P ＞ 0.05).(2) The results of ELISA showed:①) the expression of IL-1β inside the HepG2 cells in the blank control group,full-length HBx protein group,HBx1-127 group,HBx1-101 group and LPS + ATP group was (87 ± 9)pg/mL,(587 ±56)pg/mL,(125 ±12) pg/mL,(113 ± 13) pg/mL and (677 ± 74) pg/mL,respectively,with a statistically significant difference among the 5 groups (F =139.010,P ＜ 0.05).The expression of IL-1 β of LPS + ATP group was significantly different from that of blank control group,HBx1-127 group and HBx1-101 group (t =13.691,12.752,13.001,P ＜0.05).The expression of IL-1β of full-length HBx group was significantly different from that of the HBx1-127 group and the HBx1-101 group (t =14.051,14.283,P ＜ 0.05).There was no statistically significant difference between the LPS + ATP group and the full-length HBx protein group (t =1.691,P ＞0.05).The expression of IL-18 in the blank control group,full-length HBx protein group,HBx1-127 group,HBx1-101 group and LPS + ATP group was (43 ±8)pg/mL,(252 ±38)pg/mL,(70 ± 13)pg/mL,(63 ± 10)pg/mL and (263 ±48)pg/mL,respectively,with a statistically significant difference among the 5 groups (F =44.010,P ＜0.05).The expression of IL-18 of LPS + ATP group was significantly different from that of blank control group,HBx1-127 group and HBx1-101 group,respectively (t =7.848,6.722,7.065,P ＜ 0.05).The expression of IL-18 of full-length HBx group was significantly different from that of HBx1-127 group and HBx1-101 group (t =7.882,8.331,P ＜ 0.05).There was no statistically significant difference between LPS + ATP group and full-length HBx group (t =0.326,P ＞ 0.05).②The expressions of IL-1β and IL-18 in the HepG2 cells of the full-length HBx protein were (587 ± 91)pg/mL and (243 ± 22) pg/mL before the addition of glibenclamide,(115 ± 17) pg/mL and (90 ± 12) pg/mL after the addition of glibenclamide,respectively,with statistically significant differences before and after the addition of glibenclamide (t =8.800,10.566,P ＜ 0.05).The expressions of IL-1β and IL-18 in the HepG2 cells of the full-length HBx protein were (573 ± 89) pg/mL and (252 ± 24) pg/mL before the addition of APDC,(124 ±21)pg/mL and (116 ± 15)pg/mL after the addition of APDC,respectively,with statistically significant differences before and after the addition of APDC (t =8.516,8.269,P ＜ 0.05).(3) The results of flow cytometry showed that the relative expression of reactive oxygen in the HepG2 cells in blank control group,fulllength HBx protein group and LPS + ATP group were 66 ± 14,275 ± 54 and 388 ± 88,with statistically significant differences among the 3 groups (F =22.130,P ＜ 0.05) and between the full-length HBx protein group or LPS +ATP group and blank control group (t =6.489,6.256,P ＜ 0.05).There was no statistically significant difference between full-length HBx protein group and LPS + ATP group (t =1.887,P ＞ 0.05).Conclusion HBx protein may play an important role in the occurrence and development of HBV-related HCC by activating NLRP3 inflammasome through inducing reactive oxygen generation in the HepG2 cells.
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Hepatic cellular cancer (HCC) is one of the most common cancers in the world, which is a serious threat to human health and life quality. More than 700 000 people die of HCC each year on average, and its incidence increases in many countries. Chronic hepatitis B virus (HBV) infection has been identified as a dominant risk factor for HCC. The pathogenesis of HBV-induced hepatocarcinogenesis is, however, incom-pletely understood. Evidence currently available supports a key role of the HBV X protein (HBx) in the cancer transformation and malignant tumor metastasis. HBx is a multifunctional regulator that may cooperate with the host factors to exert its effects on transcription, signal transduction, cell cycle progression, apoptosis, protein degradation, expression of oncogene and anti-oncogene. This review presents the current knowledge in the molecular pathogenesis of HBx in the induction of HCC.
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Background and purpose:Hepatitis B virus X protein (HBx) and hypoxia inducible factor-1α(HIF-1α) play key roles in hepatocarcinogenesis and the development of hepatocellular carcinoma. Positive correlation on the expression of these 2 proteins in hepatocellular carcinoma tissues has been found, whereas the underlying mechanisms have not been fully elucidated. This study focused on the role of HBx in regulating HIF-1α and the underlying mechanisms in hepatocellular carcinoma cells. Methods:The expression plasmids were transfected into Huh7 cells with LipofectemineTM 2000. Western blot analysis was applied to detect the expressions of HIF-1αand HIF-1β protein. The transcriptional activity of HIF-1α was detected by the commercial analysis kits. The mRNA levels of HIF-1αand its target genes, including vascular endothelial growth factor (VEGF) and multi-drug resistance gene 1 (MDR1), were detected by quantitative real-time PCR (qRT-PCR). Immunoprecipitation analysis was applied to detect the interaction of HIF-1α, HBx and protein von Hippel-Lindau (pVHL). Results:Huh7 cells transfected with HBx plasmid led to sharp increase of HIF-1αprotein and transcriptional activity, as well as the mRNA of VEGF and MDR1 (P0.05). Meanwhile, HBx also signiifcantly impaired the function of pVHL in mediating the degradation of HIF-1αby ubiquitin hydrolase. This finding was further confirmed by the immunoprecipitation analysis, which showed that HBx could directly bind to pVHL, but not to HIF-1α. Conclusion:HBx may inhibit the inter-activation between pVHL and HIF-1αthrough directly binding to pVHL, and thus enhance the stability and transcriptional activity of HIF-1α.
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AIM: To investigate the correlation of hepatitis B virus X protein (HBx) with renal tubular epithelialcell apoptosis in hepatitis B virus-associated glomerulonephritis (HBVGN) and the possible signaling mechanism. METHODS: The activation of JAK2/STAT3 signal pathway and the expression of apoptosis -related proteins in humankindey proximal tubular epithelial cells (HK-2 cells) were determined by Western blotting after transfection with HBx eukaryoticexpression vector.The cell proliferation was observed by CCK-8 assay.The cell apoptosis was analyzed by the imagingof HO33342 staining, transmission electron microscopy and flow cytometry with Annexin V /PI double staining.RESULTS:After transfection of the target gene HBx, the expression levels of both p-JAK2 and p-STAT3 were significantly increased.At the same time, the cell proliferation was obviously inhibited, and the apoptotic rate was increased.After incubationwith AG490, the JAK2/STAT3 signal pathway was partially blocked, and the cell apoptosis induced by HBx was reduced. CONCLUSION: HBx up-regulates the activation of JAK2/STAT3 signal pathway to induce renal tubular epithelialcell apoptosis, which is possibly involved in the pathogenic mechanism that HBV directly damages nephridial tissue .
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Six transmembrane protein of prostate 2 (STAMP2) plays a key role in linking inflammatory and diet-derived signals to systemic metabolism. STAMP2 is induced by nutrients/feeding as well as by cytokines such as TNFalpha, IL-1beta, and IL-6. Here, we demonstrated that STAMP2 protein physically interacts with and decreases the stability of hepatitis B virus X protein (HBx), thereby counteracting HBx-induced hepatic lipid accumulation and insulin resistance. STAMP2 suppressed the HBx-mediated transcription of lipogenic and adipogenic genes. Furthermore, STAMP2 prevented HBx-induced degradation of IRS1 protein, which mediates hepatic insulin signaling, as well as restored insulin-mediated inhibition of gluconeogenic enzyme expression, which are gluconeogenic genes. We also demonstrated reciprocal expression of HBx and STAMP2 in HBx transgenic mice. These results suggest that hepatic STAMP2 antagonizes HBx-mediated hepatocyte dysfunction, thereby protecting hepatocytes from HBV gene expression.
Subject(s)
Animals , Female , Humans , Male , Mice , Gene Expression , Gluconeogenesis/genetics , Hep G2 Cells , Insulin/pharmacology , Insulin Receptor Substrate Proteins/genetics , Insulin Resistance , Lipid Metabolism , Liver/metabolism , Membrane Proteins/metabolism , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Transgenic , Oxidoreductases/metabolism , Phosphorylation , Protein Binding , Protein Processing, Post-Translational , Proteolysis , Receptor, Insulin/metabolism , Trans-Activators/physiology , Transcriptional ActivationABSTRACT
Hepatocellular carcinoma(HCC)shows high malignancy and metastasis. Hepatitis B virus X protein (HBx), a virus protein coded by hepatitis B virus, plays an important role in HCC metastasis. Recently, it was reported that HBx activates metastatic signal-pathways through MMPs, VEGF, uPA etc.,and promotes metastasis of hepatocellular carcinoma.
ABSTRACT
Objective To investigate the effects and possible mechanism of action of inhibiting hepatitis B virus X protein (HBx) expression on liver cancer metastasis. Methods The suppression of HBx expression in MHCC97H cells was performed by siRNA interference technique, and the effects of HBx suppression on the metastasis of MHCC97H cells were detected by Matrigel invasion assays and in a lung-metastasis mouse model. The expression levels of related epithelial-mesenchymal transition (EMT) and apoptosis proteins were examined by Western blotting. Results Introduction of HBx-siRNA into MHCC97H cells inhibited the expression of HBx and the ability to metastasize,downregulated the expression of Twist and N-cadherin, and upregulated E-cadherin expression. These changes resulted in inhibiting EMT of MHCC97H cells. Meanwhile, apoptosis involved in the Twist-P53 pathway was also found. Conclusions Inhibiting expression of HBx can decrease the metastatic a-bility of MHCC97H cells by changing EMT and inducing apoptosis.
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AIM: To construct HBx eukaryotic expression vector pEYFP-C1-X and eukaryotic expression vector pGL3-P4 driven by P4 promoter of human IGF-II gene and to investigate the effect of HBx on the transcription activity of IGF-II gene P4 promoter. METHODS: HBx gene and P4 promoters were cloned into pEYFP-C1 and pGL3-basic vectors respectively by gene recombination techniques to construct recombinant plasmids pEYFP-C1-X and pGL3-P4. HepG2 cells were transfected with pEYFP-C1-X and the resistant cell clones were selected by G418. Then methylated pGL3-P4 was transiently transfected into the above cell clones, and the transcription activity of P4 promoter was determined by dual-luciferase reporter assay system. RESULTS: (1) Aim fragments HBx gene and P4 promoter that were cloned were 465 bp and 1 246 bp, respectively and the DNA sequences were accordant with GenBank data confirmed by restricted enzyme digestion and sequencing. (2) HepG2-EYFP-X cells that expressed HBx protein were obtained. (3) Luciferase activity of methylated P4 promoter in the HepG2-EYFP-X was more than that of control cell HepG2-EYFP (P<0.01). CONCLUSION: HBx may enhance the transcription activity of the P4 promoter.
ABSTRACT
Objective:To construct subtractive cDNA library from human hepatocellular carcinoma cells transactivated by C-terminally truncated 40 amino acids using suppression subtractive hybridization(SSH) technique and to clone the associated genes.Methods: Huh-7 cells were separately transfected with pcDNA3(-) harboring the sequence of HBx protein C-terminally truncated 40 amino acids and pcDNA3(-) harboring the full length sequence of HBx protein vectors.The total RNAs were isolated from the transfected Huh-7 cells and were reversely transcripted into double strand cDNAs.After the cDNAs were digested with restriction enzyme RsaⅠ,they were divided into 2 groups and were ligated to the special adaptor 1 and adaptor 2R,respectively.The tester cDNAs were then hybridized with driver cDNAs twice and the products were amplified twice by nested PCR technique.The PCR products were connected with pUCm-T plasmid vectors to establish the subtractive library.Amplification of the library was carried out with E.coli strain JM109.The inserts of cDNAs were sequenced and analyzed in GenBank with Blast search.Results: The subtractive cDNA library was successfully constructed.The amplified library contained 154 positive clones,and colony PCR showed that these clones contained 200-800 bp inserts;some fragments coded proteins involved proto-oncogenes,cell signaling genes,cell growth factor genes,cell apoptosis genes,metabolism and protein synthesis genes.Conclusion: Subtractive cDNA library has been successfully constructed by SSH technique,which may help to clone novel genes transactivated by HBx C-terminally truncated 40 amino acids and to explore the molecular mechanism of hepatoma pathogenesis.