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@#Abstract: Objective To construct HepG2, Huh7 cell lines stably express hepatitis B virus X (HBx) mutant (C1653T, T1753C), and explore their effect on the biological behavior of hepatocellular carcinoma cells. Methods The lentivirus plasmid of pLVX-HBxC1653T-IRES-tdTomato, pLVX-HBxT1753C-IRES-tdTomato were obtained by PCR site mutagenesis according to wild type ayr HBx. Double enzyme digestion and Sanger sequencing were performed for accuracy of plasmid. Blank HepG2 and Huh7 cells were used as the control group, HepG2, Huh7 cells were infected by pLVX-HBx-IRES-tdTomato, pLVX-HBxC1653T-IRES-tdTomato, and pLVX-HBxT1753C-IRES-tdTomato lentivirus solution, then monoclonal cell was selected by 0.6 μg/mL puromycin. Immunostaining and Western Blot were performed for the verification of stable strains. CCK8 assay was performed for the proliferation capacity of stable strains. Western Blot was performed for expression of EMT-related signal molecules in cells. The independent samples t-test was used for comparison between two groups. Results Double enzyme digestion and Sanger sequencing showed that that the size of the cut fragments of recombinant lentiviral plasmids was correct, and the point mutation location and base substitution were correct, suggesting that the plasmid of pLVX-HBx-IRES-tdTomato, pLVX-HBxC1653T-IRES-tdTomato, pLVX-HBxT1753C-IRES-tdTomato were constructed successfully. Immunostaining and Western blot showed that HBX were expressed in stable strains, while there was no HBX expression in the blank control group, indicating that the HepG2 and Huh7 cell lines stably expressing HBx, HBxC1653T, HBxT1753C were successfully constructed. CCK8 assay showed that the proliferation capacity of HBx and mutant were enhanced compared to the control group (P<0.01), HBx C1653T displayed further additive the effect compared to HBx (P<0.05). Moreover, HBxC1653T mutation also significantly upregulated N-cadherin expression and downregulated E-cadherin expression, thus promoting the occurrence of EMT. Conclusions HepG2 and Huh7 cell lines stably expressing HBx, HBxC1653T, HBxT1753C were successfully constructed, HBxC1653T mutation significantly enhanced the proliferation of HCC cells and epithelial to mesenchymal transition occurrence.
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Objective@#To investigate the influence of hepatitis B virus X gene (HBx) on apoptosis of hepatic cells mediated by Fas in HePG2 cells.@*Methods@#HBx eukaryotic vector pcDNA3.1(+)-X was transfected into HEPG2 cells with lipofectamine, and the null vector pcDNA3.1(+) and untransfected HEPG2 were used as normal controls. The cells were collected 72 h after transfection, and the expression of HBx mRNA and protein was determined using RT-PCR and Western blot, respectively. The mRNA expression of apoptosis-related genes Bcl-2 and Bax mRNA was also determined using RT-PCR. Cytotoxicity and apoptosis were evaluated using CCK-8 and flow cytometry, respectively, after HepG2-HBx and HepG2-3.1 cells were treated with stimulatory monoclonal antibody anti-Fas CH11. The t test was used for pairwise comparison.@*Results@#The cell line HepG2-HBx was successfully established, as confirmed by RT-PCR and Western blot, and RT-PCR results showed that HepG2-HBx cells had significantly higher expression of Bcl-2 mRNA than HepG2-3.1 and HepG2 cells (P < 0.05), but had significantly lower expression of Bax mRNA than HepG2-3.1 and HepG2 cells (P < 0.05); CCK-8 and flow cytometry showed that anti-Fas CH11 had a lower cytotoxicity to HepG2-HBx cells and allowed for a lower apoptosis rate of HepG2-HBx cells compared with HepG2-3.1 and HepG2 cells.@*Conclusions@#HBx can inhibit apoptosis of hepatic cells mediated by the Fas pathway.
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HBx gene is a multifunctional regulator,which has extensive trans-activating effects,and can activate transcription factors,inhibit DNA repair and regulate cell proliferation,differentiation and apoptosis.In recent years,the role of HBx gene in pathogenesis of hepatitis B virus-associated glomemlonephritis (HBV-GN) has been extensively studied,and the results show that HBx can promote glomerular mesangial cell proliferation,and induce damage or apoptosis of podocytes and renal tubular epithelial cells.This paper reviews the research progress on biological characteristics of HBx and its role in pathogenesis of HBV-GN.
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Objective To investigate the effect of liver X receptor α (LXRα) gene silencing on lipid metabolism-related genes in HepG2. 2. 15 cells. Methods HepG2. 2. 15 cells were divided into blank control group (without transfection), negative control group (transfected with HK plasmid), and shLXRα group(transfected with shLXRα plasmid). The shLXRα plasmids carrying LXRα gene were constructed and were used to transfect HepG2. 2. 15 cells using Polyjet™ reagent. Green fluorescent protein and LXRα protein expression were examined by fluorescence microscope and Western blotting analysis 24-96 h after transfection, so as to identify the best interference time. Then cells were treated with agonis T090131 fo 2 o 4 h, and the content of triglyceride (TG) was observed to detect the degree of steatosis by biochemical assay. The expression of sterol regulatory element binding protein-lc (SREBP-lc) mRNA was detected by RT-PCR and the expression of hepatitis B virus X (HBx) protein and fatty acid synthase (FAS) protein was tested by Western blotting analysis. Results The shLXRa plasmid was constructed and transfected into HepG2. 2. 15 cells successfully. Compared with blank and negative control groups, LXRα protein was markedly decreased in the shLXRα group, with the lowest level found at 48-72 h after transfection (P<0. 01). Afte cell wer stimulate wit T090131, HBx and FAS protein expression, the content of TG, and SREBP-lc mRNA expression gradually increased with the prolongation of stimulation period, and there was no significant difference in HBx expression at the same time point between different groups. FAS protein, TG contents, and SREBP-lc mRNA in shLXRα group were significantly lower than those in the other two groups (P<0. 01). Conclusion HBx can regulate lipid metabolism through LXRa/SREBP-lc/FAS pathway.
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Objective:To construct eukaryotic expression vector for hepatitis B virus X(HBV X) gene with enhanced green fluorescence protein(EGFP),and confirm its expression in hepatocellular carcinoma(HCC)cell line.Method: HBV X gene was cloned from pcDNA3.1(+)-X by enzyme digestion and inserted to pEGFP-N1.The rector was confirmed by enzyme digestion,PCR assay and sequencing.Then pEGFP-X was transfected to HCC cell line Bel-7402.After transient transfection,expressions of HBV X and EGFP gene were detected by RT-PCR and fluorescence microscope respectively.Results: The results showed that the recombinant plasmid could express HBV X gene efficiently in Bel-7402,which showed green fluorescence.Conclution: The pEGFP-X was constructed and the fused HBV X-EGFP gene was expressed in Bel-7402 successfully.These facilitate the study of the effect of HBV X protein on the development of HCC.
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BACKGROUND/AIMS: Hepatitis B virus (HBV) is the etiological factor for hepatocellular carcinoma (HCC). Numerous evidence has indicated a link between chronic infection with HBV and the development of HCC. Among the four proteins encoded by HBV, Hepatitis B virus X gene(HBx), best characterized as a transcriptional transactivator, gained attention owing to its presumptive role in oncogenesis. Further, HBx has been shown to stimulate signal transduction pathways such as Ras-MAPK pathway, NF-kappaB, and Src kinase. The pleiotropic events caused by HBx may be the key to understanding the HBV-mediated oncogenicity. However, the specific roles of HBx in oncogenesis remain largely elusive. To explore the role of HBx in hepatocarcinogenesis, we examined the deregulation of host genes induced by HBx expression. METHODS: HBx was ectopically expressed in HepG2 cells using a recombinant adenovirus to transiently express HBx. Gene expression profiling of HBx was conducted on cDNA microarrays that contained 1,028 cDNAs. RESULTS: A number of oncogenes and genes that are involved in cell growth, DNA repair, cell cycle regulation, and cell motility were deregulated by HBx. CONCLUSIONS: Theses results suggest that HBx regulates transcription in a way that contributes to the proliferation of hepatocytes, a probable early event of HCC.