HepG2-HBx Cells Research Articles

HBx Downregulates miR-422a Expression via Activation of FOXG1/Q1/E1 in HepG2 Cells

microRNA-422a (miR-422a) is downregulated in many hematopoietic tumors and solid tumors including hepatocellular carcinoma. We previously demonstrated that hepatitis B virus X protein (HBx) downregulated expression of miR-422a in HCC cell line HepG2 in vitro. However, we explore the mechanisms underlying this action. Forkhead box proteins (FOX) G1/Q1/E1 are known to negatively regulate miR-422a expression, and this prompted us to determine whether HBx suppresses miR-422a expression via activation of FOXG1/Q1/E1. The relationship between FOXG1/Q1/E1 and miR-422a in HepG2 cells stably expressing HBx was assessed with qRT-PCR. The correlation between HBx and FOXG1/Q1/E1 was determined with qRT-PCR and western blot in vitro. The cell cycle and CCK-8 assays were used to elucidate the consequence of miR-422a transfection in HepG2-hbx cells. FOXG1/Q1/E1 activated by HBx was found to be responsible, at least in part, for the downregulation of miR-422a in HepG2 cells. miR-422a transfection hampered the growth of HepG2-hbx cells by arresting cells in G1 phase. Both FOXG1/Q1/E1 and miR-422a may be suitable molecular targets for treatment of HBV-infected HCC.

Hepatitis B Virus X Protein Induces SATB1 Expression Through Activation of ERK and p38MAPK Pathways to Suppress Anoikis.

We previously reported that there were potentially certain correlations between the high expression of SATB1 and the HBV infection in human hepatocellular carcinoma tissues, and SATB1 promoted tumor growth and metastasis in liver cancer. Hepatitis B virus (HBV) infection is internationally recognized as a contributing factor to metastasis in liver cancer. The anoikis prevention of detached malignant cancer cells is the precondition for metastasis. Our studies aimed to explore the relationship between HBV infection, SATB1 and liver cancer cell anoikis and their specific regulatory mechanisms in HBV-associated liver cancer. HepG2 cell was transiently transfected with pBlue-HBV and seven types of HBV-encoded protein plasmids. Anoikis assay and soft agarose colony formation experiment were analyzed in HepG2.2.15-SATB1 siRNA cells, HBx-overexpressing cells and HepG2-HBx-SATB1 siRNA cells. The inhibitors of signaling molecules were used to treat of HepG2-HBx cells, and then, the SATB1 expression and phosphorylation levels of signaling molecules were evaluated. Our data show that the high expression of SATB1 and enhanced anoikis resistance were observed in HBV stably expressing cell line HepG2.2.15 and high metastatic potential cell line SK-HEP-1. HBV can induce SATB1 expression and suppress anoikis of unattached liver cancer cells. Moreover, SATB1 expression and anoikis resistance were mainly regulated by HBV-encoded viral protein HBx through the activation of ERK and p38 MAPK signaling pathways to promote metastasis of liver cancer. These data suggest that the HBV-encoded HBx and SATB1 may play an important role in promoting anoikis resistance and metastasis in HBV-associated liver cancer.

Compound Phyllanthus urinaria L Inhibits HBV-Related HCC through HBx-SHH Pathway Axis Inactivation.

Compound Phyllanthus urinaria L (CP) is a traditional formula widely used in clinical practice for hepatocellular carcinoma (HCC), especially HBV-related HCC. HBx, HBV X gene encoded X protein, has positive correlation with the abnormal SHH pathway in HBV-related HCC. So, we predicted that CP has the capability of anti-HBV-related HCC maybe via inactivating the HBx-Hedgehog pathway axis. HepG2-HBx cells, HBx overexpression, were treated with CP (70μg/ml and 35 μg/ml, respectively) for 48 hours and the mice which received the HepG2-HBx cells were treated with CP (625mg/kg and 300 mg/kg, respectively) for 17 days to evaluate the effect of CP on HBV-related HCC. HBx could accelerate HepG2 cells proliferation, clone formation, and migration in vitro and also could strengthen tumor growth in mice. However, CP could significantly decrease HepG2-HBx cells proliferation, clone formation, and migration in vitro and also could inhibit tumors growth in mice in a dose-dependent manner. Mechanism studies suggested that HBx upregulated the mRNA and proteins expression of Sonic hedgehog (SHH), transmembrane receptor patched (PTCH-1), smoothened (SMO), oncogene homolog transcription factors-1 (GLI-1), and oncogene homolog transcription factors-2 (GLI-2), which are compositions of the SHH pathway. CP could inhibit the mRNA and proteins expression of SHH, PTCH-1, GLI-1, and HBx. It may be one of the underlying mechanisms of CP to delay the HBV-related HCC development through the HBx-SHH pathway axis inactivation.

Influence of hepatitis B virus X gene on apoptosis of hepatic cells mediated by Fas

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.

Downregulation of mPGES‐1 Expression via EGR1 Plays an Important Role in Inhibition of Caffeine on PGE2 Synthesis of HBx(+) Hepatocytes

We investigated the mechanism of caffeine in influencing HBx(+) hepatocytes to synthesize PGE2. The inhibitory effect of caffeine on hepatocyte proliferation increased with increasing caffeine concentrations (200–800 μM) and treatment times (1–7 days), which was first observed at the second test time point (caffeine treatment for 4 days). The inhibition of caffeine on the growth of HL7702-HBx and HepG2-HBx cells was most obvious at 800 μM caffeine and at caffeine treatment for 7 days. The PGE2 secretion and the expression of mPGES-1 and EGR1 were downregulated, whereas PPARγ expression was upregulated. The mPGES-1 promoter activity of HBx(+) hepatocytes decreased more significantly than that of HBx(−) hepatocytes. Moreover, the expression of EGR1 and PPARγ changed more significantly in HBx(+) hepatocytes cultured for 12 to 24 hours in the presence of 5 mM caffeine. This limited success may be attributed to caffeine releasing the binding of HBx and PPARγ and furthermore affecting the mPGES-1 expression by EGR1 in HBx(+) hepatocytes. The results indicate that caffeine could effectively reduce PGE2 synthesis in HBx(+) hepatocytes by specifically blocking the PPARγ-EGR1-mPGES-1 pathway, thereby providing a new evidence of molecular biology for the hypothesis that drinking coffee is beneficial to HBV-infected patients.

Hepatitis B virus X protein promotes insulin-like growth factor II gene expression by inducing hypomethylation of the P3 promoter in hepatocellular carcinoma

To explore the involvement of hepatitis B X protein (HBx) in promoter 3 (P3)-driven mRNA overexpression of the insulin-like growth factor II gene (IGF-II) and investigate the underlying epigenetic mechanism. Levels of P3 and HBx mRNA and status of P3 methylation were analyzed in human hepatocellular carcinoma (HCC) samples, with and without hepatitis B virus (HBV) infection, using quantitative reverse transcription-PCR and bisulfite sequencing. In addition, the levels of P3 mRNA and P3 methylation were examined in HepG2 cells stably overexpressing HBx (HepG2-HBx). Finally, P3 promoter-luciferase constructs were cotransfected into HepG2 cells along with an HBx-expressing plasmid, and the effects of HBx on transcriptional activity and methylation of P3 were analyzed. Statistical analyses of the data were conducted by chi square test, Fisher's exact test, Student's t-test, Marn-Whitney U test, and Pearson's correlation coefficient test. The HBV-positive HCC specimens had significantly higher levels of P3 mRNA than the HBV-negative HCC specimens (-9.59 ± 3.22 vs. -12.97 ± 3.08 delta CT; P=0.006) but significantly lower levels of P3 methylation (mean values for the 17 CpG sites (36.9% ± 15.5% vs. 52.1% ± 19.1%; P=0.025). The P3 transcript abundance was positively correlated with the level of HBx expression and negatively correlated with the level of P3 methylation. The epigenetic results from experiments with the HepG2-HBx cells were similar. Transfection of HBx significantly decreased P3 methylation level and increased its activity. HBx expression may promote IGF-II expression by inducing hypomethylation of its P3 promoter in hepatocellular carcinoma.

Hepatitis B virus X protein promotes P3 transcript expression of the insulin‐like growth factor 2 gene via inducing hypomethylation of P3 promoter in hepatocellular carcinoma

Hepatitis B virus (HBV) X protein (HBx) contributes to hepatocarcinogenesis. The overexpression of transcripts from P3 and P4 promoters of the insulin-like growth factor 2 (IGF2) gene is observed in hepatocellular carcinoma (HCC). Here, we aimed to explore the involvement of HBx in P3-driven mRNA overexpression and underlying epigenetic mechanism. P3 mRNA, P3 methylation status, HBx mRNA and HBx protein were analysed in human HCC samples with and without HBV infection using quantitative RT-PCR, bisulphite sequencing and Western blotting. The effects of HBx on P3 mRNA expression, and P3 transcriptional activity and methylation were further evaluated in HCC cell lines. P3 mRNA level was higher and P3 methylation level was lower in HBV-positive HCC specimens compared with those of HBV-negative HCC specimens. P3 transcript abundance was positively correlated with HBx expression and negatively correlated with P3 methylation in HCC specimens. The stable expression of HBx upregulated P3 mRNA expression and reduced P3 methylation level in HepG2-HBx cells. The transient expression of HBx stimulated P3 promoter activity and decreased P3 methylation level of P3 promoter-luciferase construct in a dose-dependent manner in HepG2 and Huh-7 cells. Furthermore, HBx mRNA expression was found to be independent predictive factors for both shorter disease-free survival time and shorter overall survival time of HCC patients. HBx may promote IGF2-P3 transcript expression by inducing hypomethylation of P3 promoter and may be associated with an inferior clinical outcome of HBV-related HCC patients. This study provides useful information for understanding the mechanism of HBx-mediated HCC.

INOS promotes HBx-induced hepatocellular carcinoma via upregulation of JNK activation

Inducible nitric oxide (iNOS) is closely correlated with chronic inflammation in hepatitis B virus X protein (HBx)-induced hepatocellular carcinoma (HCC). However, the molecular mechanisms through which iNOS contribute to hepatocarcinogenesis remain poorly understood. Therefore, we investigated the role of iNOS in signaling pathways underlying HBx-induced liver tumorigenesis. iNOS deletion showed a marked decrease in the hepatic tumor size and stage of HBx transgenic (Tg) mice, indicating a strong contribution of iNOS signaling pathways to hepatocarcinogenesis. In addition, we found that nitric oxide (NO) increased HBx mRNA by recruiting CREB to the CRE site of HBV enhancer in HepG2 cells, suggesting a positive feedback loop between HBx and iNOS signaling pathway. Moreover, iNOS-modulated JNK activation was associated with sustained upregulation of Cyclin D1 in HBxTg mice and HepG2-HBx cells. These results imply that iNOS may play a key role in HBx-associated HCC development. Taken together, our findings demonstrate that iNOS aligns with HBx to promote tumor progression. These findings provide a better understating of the mechanism involving HBx-mediated hepatic tumorigenesis and selective inhibition of iNOS may have therapeutic applications in HBx-associated HCC.

Role of hepatitis B virus X protein in regulating LIM and SH3 protein 1 (LASP-1) expression to mediate proliferation and migration of hepatoma cells

BackgroundHepatitis B virus X protein (HBx) has been shown to be responsible for the development of hepatocellular carcinoma (HCC) caused by Hepatitis B virus infection. However, its potential effect on the progression of hepatocellular carcinoma remains yet unclear. LIM and SH3 protein 1 (LASP-1), a focal adhesion protein, is expressed in an up-regulation manner in the HCC tissues. LASP-1 plays an important role in the regulation of proliferation and migration of HCC. In this study, we investigated the effect of LASP-1 involved in HBx-related tumor progression.MethodsLASP-1 levels in the HBx stable transfected HepG2 and Huh-7 cells were detected by RT-PCR and western blot analysis. The cellular localization of LASP-1 was assessed by immunofluorescence analysis. The activity of phosphatidylinositol 3-kinase (PI3-K) pathway was demonstrated by western blot assay. The HBx-expressing cells were transfected with specific small interference RNA (siRNA) against LASP-1. The proliferation and migration ability of cells were evaluated by cell viability assay and plate clone formation assay. The migration ability of cells was detected by transwell assay and wound healing assay.ResultsRT-PCR and western blot analysis indicated the expression of LASP-1 was increased in the stable HBx-expressing cells compared with the control cells. Immunofluorescence study revealed that the distributions of LASP-1 in HepG2-HBX cells were mainly in pseudopods and the cytoplasm while they were mainly localized in the cytoplasm of HepG2-Mock cells. The cellular localizations of LASP-1 in Huh-7-HBX cells were in the perinuclear fractions while they were mainly localized in the cytoplasm of Huh-7-Mock cells. The upregulation of LASP-1 was inhibited after treatment with LY294002, PI3-K pathway inhibitor. Overexpression of LASP-1 in the stable HBx-expressing cells enhanced the proliferation and migration ability of hepatocellular cells. siRNA-mediated LASP-1 knowdown in the stable HBx-expressing cells significantly suppressed hepatocellular cells proliferation and migration.ConclusionsThese results demonstrated that HBx could upregulate LASP-1 through PI3-K pathway to promote the proliferation and migration of hepatoma cells.

Hepatitis B Virus X Protein Regulates Hepatic Glucose Homeostasis via Activation of Inducible Nitric Oxide Synthase

Dysregulation of liver functions leads to insulin resistance causing type 2 diabetes mellitus and is often found in chronic liver diseases. However, the mechanisms of hepatic dysfunction leading to hepatic metabolic disorder are still poorly understood in chronic liver diseases. The current work investigated the role of hepatitis B virus X protein (HBx) in regulating glucose metabolism. We studied HBx-overexpressing (HBxTg) mice and HBxTg mice lacking inducible nitric oxide synthase (iNOS). Here we show that gene expressions of the key gluconeogenic enzymes were significantly increased in HepG2 cells expressing HBx (HepG2-HBx) and in non-tumor liver tissues of hepatitis B virus patients with high levels of HBx expression. In the liver of HBxTg mice, the expressions of gluconeogenic genes were also elevated, leading to hyperglycemia by increasing hepatic glucose production. However, this effect was insufficient to cause systemic insulin resistance. Importantly, the actions of HBx on hepatic glucose metabolism are thought to be mediated via iNOS signaling, as evidenced by the fact that deficiency of iNOS restored HBx-induced hyperglycemia by suppressing the gene expression of gluconeogenic enzymes. Treatment of HepG2-HBx cells with nitric oxide (NO) caused a significant increase in the expression of gluconeogenic genes, but JNK1 inhibition was completely normalized. Furthermore, hyperactivation of JNK1 in the liver of HBxTg mice was also suppressed in the absence of iNOS, indicating the critical role for JNK in the mutual regulation of HBx- and iNOS-mediated glucose metabolism. These findings establish a novel mechanism of HBx-driven hepatic metabolic disorder that is modulated by iNOS-mediated activation of JNK.

The Role of NF-κB in Hepatitis B Virus X Protein-Mediated Upregulation of VEGF and MMPs

ABSTRACTHepatitis B virus X protein (HBx) promotes hepatocellular carcinoma (HCC) invasion and metastasis by a poorly understood mechanism. This study investigated the role of NF-κ B in HBx-mediated upregulation of vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs). In a stably expressing HBx cell line, NF-κ B level was examined by laser scanning confocal microscopy before and after treatment with pyrrolidine dithiocarbamate (PDTC; an NF-κ B inhibitor). VEGF, MMP2, MMP9, and MMP14 mRNA and protein levels were quantitated by real-time PCR and Western blotting, respectively. HBx stimulated NF-κ B signaling and increased VEGF, MMP2, MMP9, and MMP14 mRNA and protein levels. PDTC treatment blocked HBx-mediated stimulation of NF-κ B signaling and decreased VEGF, MMP9, and MMP14 (but not MMP2) mRNA and protein levels. In vivo studies, PDTC reduced angiogenesis in subcutaneous xenograft of nude mice which injected HepG2-HBx cells. This suggests that NF-κ B is involved in upregulating these genes and in the HBx-mediated invasion and metastasis of HCC.

Proteomic Profiling Identifies Aberrant Epigenetic Modifications Induced by Hepatitis B Virus X Protein

The hepatitis B virus-encoded X (HBx) protein coactivates transcription of a variety of viral and cellular genes and it is believed to play essential roles in viral replication and hepatocarcinogenesis. To examine the pleiotropic effects of HBx protein on host cell protein expression, we utilized 2-DE and MS analysis to compare and identify differentially expressed proteins between a stable HBx-transfected cell line (HepG2-HBx), constitutively expressing HBx, and vector control cells. Of the 60 spots identified as differentially expressed (+/- over 2-fold, p < 0.05) between the two cell lines, 54 spots were positively identified by MS/MS analysis. Several recent studies suggested that HBx was involved in regional hypermethylation of tumor suppressor genes and global hypomethylation of satellite 2 repeats during hepatocarcinogenesis; however, no specific gene has been reported as hypomethylated by HBx. Promoter methylation analysis was examined for those protein spots showing significant alterations, and our results revealed that specific genes, such as aldehyde dehydrogenase 1 (ALDH1), can be hypomethylated by HBx, and two calcium ion-binding proteins, S100A6 and S100A4, were hypermethylated by HBx and could be re-expressed by AZA (DNA methylase inhibitor) treatment. Moreover, via cluster and pathway analysis, we proposed a hypothetical model for the HBx regulatory circuit involving aberrant methylation of retinol metabolism-related genes and calcium homeostasis-related genes. In summary, we profiled proteome alterations between HepG2-HBx and control cells, and found that HBx not only induces regional hypermethylation but also specific hypomethylation of host cell genes.

Increased chromosomal alterations and micronuclei formation in human hepatoma HepG2 cells transfected with the hepatitis B virus HBX gene

The protein encoded by the hepatitis B virus (HBV)-X gene, HBX, has been implicated to be involved in the development of HBV-associated liver cancer. HBX is a multifunctional regulatory protein that has been identified as a potential oncogene but its exact function remains unclear. HBX was documented to interact with several factors involved in cellular DNA repair as well as compromise the cell’s ability to repair damaged DNA. We previously documented an accumulation of genetic alterations in two HepG2 cell lines independently transfected with HBV. In this report, we investigate the effect of the HBV-X gene (HBX) on the stability of the host genome using HepG2 stable transfectants (HepG2-HBX) and vector controls (HepG2-neo). We document that all HepG2-HBX clones analyzed contain HBX gene integrated and HBX transcript. Our data demonstrate that HepG2-HBX cells have an increased number of chromosome alterations and micronuclei formation compared to vector controls. A total of 10 de novo chromosomal rearrangements involving nine different chromosomes were detected in the HepG2-HBX clones, while no new rearrangements were found in vector controls. Each HepG2-HBX clone contained independently occurring de novo alterations not found in other HBX or vector clones. A three-fold increase of micronuclei formation was detected in HepG2-HBX cells compared to vector controls. Micronuclei originated from all chromosomes, however, preliminary data indicated that micronuclei originating from chromosomes 2, 3, 7, 18 and 20 were found in a greater amount in cells expressing the HBX gene. Interestingly, chromosomes 2, 18 and 20 were three of the chromosomes found rearranged in HepG2-HBX clones. These data provide evidence that genomic integrity was affected in cells expressing the HBX gene. De novo cytogenetic alterations identified in HepG2-HBX clones implicate the involvement of HBX in the process and support the hypothesis that HBX may interfere with normal cellular processes responsible for genomic integrity, increasing the risk for acquiring genetic mutations in infected hepatocytes.