HBV-associated Hepatocellular Carcinoma Tissues Research Articles

Hepatitis B Virus X Protein Represses Expression of Tumor Suppressor PTPN18 in Hepatocellular Carcinoma.

HBV-associated hepatocellular carcinoma (HCC) represents the prevalent form of HCC, with HBx protein being a crucial oncoprotein. Numerous members of the protein tyrosine phosphatase nonreceptor (PTPN) family have been confirmed to be significantly associated with the occurrence and progression of malignant tumors. Our group previously identified the involvement of PTPN13 in HCC. However, the roles of other PTPNs in HCC require further investigation. In this study, we found that PTPN18 expression was significantly downregulated within HCC tissues compared with adjacent nontumor and reference liver tissues. Functionally, PTPN18 exerted inhibitory effects on the proliferation, migration, invasion, and sphere-forming capability of HCC cells while concurrently promoting apoptotic processes. Through phospho-protein microarray screening followed by subsequent validation experiments, we identified that PTPN18 could activate the p53 signaling pathway and suppress the AKT/FOXO1 signaling cascade in HCC cells. Moreover, the HBx protein mediated the repression of PTPN18 expression by upregulating miR-128-3p. Collectively, our study unveiled the role of PTPN18 as a tumor suppressor in HBV-related HCC. Implications: Our findings revealed that PTPN18 might be a potential diagnostic and therapeutic target for HBV-related HCC.

NAMPT promotes the malignant progression of HBV-associated hepatocellular carcinoma through activation of SREBP1-mediated lipogenesis.

Metabolic reprogramming is a hallmark of cancer. The nicotinamide phosphoribosyltransferase (NAMPT)-mediated salvage pathway maintains sufficient cellular NAD levels and is required for tumorigenesis and development. However, the molecular mechanism by which NAMPT contributes to HBV-associated hepatocellular carcinoma (HCC) remains not fully understood. In the present study, our results showed that NAMPT protein was obviously upregulated in HBV-positive HCC tissues compared with HBV-negative HCC tissues. NAMPT was positively associated with aggressive HCC phenotypes and poor prognosis in HBV-positive HCC patients. NAMPT overexpression strengthened the proliferative, migratory, and invasive capacities of HBV-associated HCC cells, while NAMPT-insufficient HCC cells exhibited decreased growth and mobility. Mechanistically, we demonstrated that NAMPT activated SREBP1 (sterol regulatory element-binding protein 1) by increasing the expression and nuclear translocation of SREBP1, leading to the transcription of SREBP1 downstream lipogenesis-related genes and the production of intracellular lipids and cholesterol. Altogether, our data uncovered an important molecular mechanism by which NAMPT promoted HBV-induced HCC progression through the activation of SREBP1-triggered lipid metabolism reprogramming and suggested NAMPT as a promising prognostic biomarker and therapeutic target for HBV-associated HCC patients.

Large HBV Surface Protein-Induced Unfolded Protein Response Dynamically Regulates p27 Degradation in Hepatocellular Carcinoma Progression.

Up to 50% of hepatocellular carcinoma (HCC) is caused by hepatitis B virus (HBV) infection, and the surface protein of HBV is essential for the progression of HBV-related HCC. The expression of large HBV surface antigen (LHB) is presented in HBV-associated HCC tissues and is significantly associated with the development of HCC. Gene set enrichment analysis revealed that LHB overexpression regulates the cell cycle process. Excess LHB in HCC cells induced chronic endoplasmic reticulum (ER) stress and was significantly correlated with tumor growth in vivo. Cell cycle analysis showed that cell cycle progression from G1 to S phase was greatly enhanced in vitro. We identified intensive crosstalk between ER stress and cell cycle progression in HCC. As an important regulator of the G1/S checkpoint, p27 was transcriptionally upregulated by transcription factors ATF4 and XBP1s, downstream of the unfolded protein response pathway. Moreover, LHB-induced ER stress promoted internal ribosome-entry-site-mediated selective translation of p27, and E3 ubiquitin ligase HRD1-mediated p27 ubiquitination and degradation. Ultimately, the decrease in p27 protein levels reduced G1/S arrest and promoted the progress of HCC by regulating the cell cycle.

Canonical WNT Signaling Activated by WNT7B Contributes to L-HBs-Mediated Sorafenib Resistance in Hepatocellular Carcinoma by Inhibiting Mitophagy

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death globally, with hepatitis B virus (HBV) infection accounting for over half of all cases. HBV leads to the development of HCC according to a body of literature. Our previous research and other studies also suggest that HBV causes chemotherapeutic treatment resistance, however, the mechanism is uncertain. The WNT family, which encodes secreted signaling molecules, has been linked to carcinogenesis in a variety of malignancies, including HCC. However, little is known regarding WNT7B, a WNT ligand, in the development of HCC and HBV-induced chemoresistance. In this study, the bioinformatics analysis and immunohistochemistry (IHC) staining of clinical samples revealed that WNT7B was overexpressed in HBV-associated HCC tissues versus nontumor liver tissues, which was related to HCC patient survival. Further study in vitro showed that WNT7B and its receptor frizzled-4 (FZD4) were upregulated in response to large hepatitis B surface antigens (L-HBs). L-HBs increased canonical WNT signaling in HCC cells through WNT7B/FZD4. According to functional experiments, WNT7B enhanced the cell proliferation and metastasis in HCC. In vivo and in vitro studies investigated whether L-HBs induced sorafenib resistance by WNT7B in HCC. Interestingly, L-HBs suppressed sorafenib-induced mitophagy by increasing WNT7B/CTNNB1 signaling, resulting in chemoresistance. The findings revealed that WNT7B could be a promising molecular therapeutic target as well as a predictor of sorafenib resistance in HBV-related HCC. The suppression of HBV structural proteins such as L-HBs may play a crucial role in systemic chemotherapy resistance in HBV-associated HCC.

Screening and Identification of Key Genes in Hepatitis B Virus-Related Hepatocellular Carcinoma Through an Integrated Bioinformatics Approach

Objective: Primary liver cancer is one of the main causes of cancer mortality globally, with hepatocellular carcinoma (HCC) being the most frequent type. Chronic hepatitis B virus (HBV) infection is leading cause of HCC. This study aimed to identify significant genes for predicting prognosis in HBV-associated HCC. Methods: The GSE121248 gene expression profile was obtained from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) for HBV-associated HCC were identified by analyzing this expression profile. Enrichment analyses were performed to discover the role of DEGs in biological processes, cell components, molecular functions, and pathways. Then, protein-protein interaction (PPI) was constructed and 5 hub genes were identified. Finally, survival analysis was conducted to validate the prognostic value of these genes. Results: A total of 20188 official gene symbols were found, and 119 DEGs were identified between HBV-associated HCC and normal liver tissues. The PPI network identified CCNB1, CDK1, TOP2A, RACGAP1, and ASPM as hub genes. Kaplan-Meier curves showed that the high expression of the hub genes had significantly lower survival. Conclusion: CCNB1, CDK1, TOP2A, RACGAP1, and ASPM could be potential prognostic biomarkers and therapeutic targets for HBV-associated HCC.

Identification of genes predicting unfavorable prognosis in hepatitis B virus-associated hepatocellular carcinoma.

BackgroundTo identify potential key genes predicting unfavorable prognosis in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC).MethodsGene expression profiles of GSE121248, GSE62232, and GSE55092 from the GEO database were obtained and analyzed. Differentially expressed genes (DEGs) between HBV-associated HCC tissues and adjacent normal tissues were screened by the limma package and Venn diagram software. Functional assessment of DEGs was performed by Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). Hub genes were selected by the protein-protein interaction (PPI) network and further validated by GSE14520 clinical data.ResultsA total of 26 up-regulated genes and 76 down-regulated genes were identified by analyzing three databases. GO and KEGG analysis demonstrated that these genes were involved in cell division, metabolism-related biological processes, the p53 pathway, and the cell cycle, among others. PPI network suggested that 14 hub DEGs (TOP2A, HMMR, DTL, CCNB1, NEK2, PBK, RACGAP1, PRC1, CDK1, RRM2, ECT2, BUB1B, ANLN, and ASPM) were most dysregulated and had potential to distinguish between HBV-associated HCC and noncancerous tissues. Further survival analysis of hub genes demonstrated that high expression of TOP2A was significantly associated with poor clinical outcomes of HBV-associated HCC.ConclusionsTOP2A might serve as a key gene for prognosis and as a therapeutic target for HBV-associated HCC.

Autophagy related protein 9A increase in hepatitis B virus-associated hepatocellular carcinoma and the role in apoptosis.

The majority of hepatocellular carcinoma (HCC) cases are associated with the hepatitis B virus (HBV) infection. Autophagy related protein 9A (ATG9A) is a transmembrane protein required for autophagosome formation. In order to investigate the role of ATG9A in HBV-associated HCC, ATG9A protein expression was determined in tumor liver tissues and compared with adjacent nontumor tissues from HCC patients with or without HBV infection. In HBV-associated HCC tissues, ATG9A protein level was increased in tumor liver tissues, but not in cases of non-HBV HCC. Our findings suggested that ATG9A might be involved in HBV and cancer cell survival. Therefore, we aimed to analyze the function of ATG9A in HBV replication using RNA interference to evaluate the HBV DNA level using real-time PCR. In the present study, there were no significant differences between shATG9A-transfected HepG2.2.15 cells and the mock control. However, we found that silencing ATG9A affected apoptosis in HepG2.2.15 and HepG2 cell lines. Our results indicated that ATG9A might be partly involved in the survival of HCC. Thus, the inhibition of ATG9A together with other targets might be a potential drug target for HCC treatment.

Hepatitis B virus core protein promotes the expression of neuraminidase 1 to facilitate hepatocarcinogenesis

AbstractNeuraminidase 1 (NEU1) has been reported to be associated with hepatocellular carcinoma (HCC). However, the function and associated molecular mechanisms of NEU1 in hepatitis B virus (HBV)-related HCC have not been well investigated. In the present study, the expression of NEU1 mediated by HBV and HBV core protein (HBc) was measured in hepatoma cells. The expression of NEU1 protein was detected via immunohistochemical analysis in HBV-associated HCC tissues. The role of NEU1 in the activation of signaling pathways and epithelial-mesenchymal transition (EMT) and the proliferation and migration of hepatoma cells mediated by HBc was assessed. We found that NEU1 was upregulated in HBV-positive hepatoma cells and HBV-related HCC tissues. HBV promoted NEU1 expression at the mRNA and protein level via HBc in hepatoma cells. Mechanistically, HBc was able to enhance the activity of the NEU1 promoter through NF-κB binding sites. In addition, through the increase in NEU1 expression, HBc contributed to activation of downstream signaling pathways and EMT in hepatoma cells. Moreover, NEU1 facilitated the proliferation and migration of hepatoma cells mediated by HBc. Taken together, our findings provide novel insight into the molecular mechanism underlying the oncogenesis mediated by HBc and demonstrate that NEU1 plays a vital role in HBc-mediated functional abnormality in HCC. Thus, NEU1 may serve as a potential therapeutic target in HBV-associated HCC.

MicroRNA-802 induces hepatitis B virus replication and replication through regulating SMARCE1 expression in hepatocellular carcinoma

Growing evidences have indicated that microRNAs (miRNAs) can regulate hepatitis B virus (HBV) expression and replication, playing crucial roles in the development of HBV infection. Until now, the functional role and mechanism of miR-802 in HBV replication and expression remain unknown. We indicated that miR-802 expression was upregulated in the HBV-associated hepatocellular carcinoma (HCC) tissues compared with the adjacent noncancerous samples. In addition, we showed that the SMARCE1 expression level was downregulated in the HBV-associated HCC tissues compared with the adjacent noncancerous samples. miR-802 expression was negatively related with MARCE1 expression in HBV-associated HCC tissues. Moreover, miR-802 expression was upregulated, and SMARCE1 expression was downregulated in the HBV-infected HepG2.2.15 cells. Ectopic expression of miR-802 significantly enhanced HBV DNA replication, while knockdown of miR-802 significantly decreased HBV DNA replication. We showed that overexpression of miR-802 promoted HbsAg and HbeAg expression, while inhibition of miR-802 decreased HbsAg and HbeAg expression. Furthermore, we indicated that ectopic expression of SMARCE1 suppressed HBV DNA replication and decreased the expression level of HbsAg and HbeAg. Finally, we showed that overexpression of miR-802 promoted HBV DNA replication through regulating SMARCE1 expression. These results suggested the important roles of miR-802 on HBV expression and replication, which may shed new light on the development of treatment for HBV.

MicroRNA-1271 functions as a potential tumor suppressor in hepatitis B virus-associated hepatocellular carcinoma through the AMPK signaling pathway by binding to CCNA1.

Hepatocellular carcinoma (HCC) is mainly associated with hepatitis B virus (HBV) infection and characterized by metastasizing and infiltrating adjacent and distant tissues. Notably, microRNA-1271 (miR-1271) is a tumor suppressor in various cancers. Therefore, we evaluate the ability of miR-1271 to influence cell proliferation, migration, invasion, and apoptosis in HBV-associated HCC through the Adenosine monophosphate-activated protein kinase (AMPK) signaling pathway via targeting CCNA1. HBV-associated HCC and adjacent normal tissues were collected to identify the expression of miR-1271 and CCNA1. To verify the relationship between miR-1271 and CCNA1, we used bioinformatics prediction and the dual-luciferase reporter gene assay. The effects of miR-1271 on HBV-associated HCC cell behaviors were investigated by treatment of the miR-1271 mimic, the miR-1271 inhibitor, or small interfering RNAagainst CCNA1. The HBV-DNA quantitative assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromid assay, scratch test, transwell assay, and flow cytometry were used to detect HBV-DNA replication, cell proliferation, invasion, migration, and apoptosis. MiR-1271 showed a low expression, whereas CCNA1 showed a high expression in HBV-associated HCC tissues. We identified that miR-1271 targeted and negatively regulated CCNA1. Upregulated miR-1271 and downregulated CCNA1 inhibited the HBV-associated HCC cell HBV-DNA replication, proliferation, migration, and invasion, while accelerating apoptosis by activating the AMPK signaling pathway. MiR-1271 promotes the activation of the AMPK signaling pathway by binding to CCNA1, whereby miR-1271 suppresses HBV-associated HCC progression. This study points to a potential therapeutic approach of downregulation of miR-1271 in HBV-associated HCC treatment.

Identification of long noncoding RNAs biomarkers in patients with hepatitis B virus-associated hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common and aggressive cancer worldwide and chronic infection of hepatitis B virus (HBV) serve as one of leading causes of HCC. This study aimed to identify the novel long noncoding RNAs (lncRNAs) biomarkers for HBV-associated HCC. The lncRNA and mRNA expression profiles of HCC patients with HBV infection were downloaded from The Cancer Genome Atlas. The differentially expressed lncRNAs (DElncRNAs) and mRNAs (DEmRNAs) between HCC and adjacent tissues were identified. The optimal diagnostic and prognostic lncRNA biomarkers for HCC were identified by using feature selection procedure and classification model. Functional annotation of DEmRNAs co-expressed with these lncRNAs biomarkers were performed. Receiver operating characteristic (ROC) curve and survival analysis of these lncRNAs biomarkers were performed. qRT-PCR validation was performed. A total of 82DElncRNAs and 805DEmRNAs between HBV-associated HCC and normal tissues were identified. CAPN10-AS1, LINC01093, RP5-890E16.2, FENDRR and C17orf82 were selected as optimal diagnostic and prognostic lncRNA biomarkers for HBV-associated HCC that were co-expressed with 105, 86, 70, 30 and 1DEmRNAs, respectively. Based on the DEmRNAs co-expressed with these five lncRNAs biomarkers, Jak-STAT signaling pathway and retinol metabolism were two significantly enriched pathways. The result in qRT-PCR validation were consistent with our analysis based on TCGA, generally. This study identified five potential lncRNAs biomarkers for HBV-associated HCC with great diagnostic and prognostic value and provided clues for their functions in HBV-associated HCC.

Reduced HBV cccDNA and HBsAg in HBV-associated hepatocellular carcinoma tissues

Approximately 50% of hepatocellular carcinoma (HCC) is attributable to chronic infection with hepatitis B virus (HBV). Serum hepatitis B surface antigen (HBsAg) is an important diagnostic marker of HBV infection, whereas intrahepatic HBV covalently closed circular DNA (cccDNA) is a surrogate marker of HBV persistence. This study aimed to investigate relationships between serum HBsAg, intrahepatic HBsAg, and intrahepatic cccDNA in HBV-associated HCC. Intrahepatic HBsAg was determined by immunohistochemistry in matched non-cancerous and HCC tissues from 88 patients; 56 patients (63.64%) were serum HBsAg positive. In serum HBsAg-positive group, intrahepatic HBsAg was positive staining in 73.2% of non-cancerous tissues, but only in 10.7% of HCC tissues. Significant correlation between serum HBsAg and intrahepatic HBsAg was observed in non-cancerous tissues (p < 0.001), but not in HCC tissues (p = 0.415). Absolute quantification of intrahepatic cccDNA was performed by droplet digital PCR in tissues from 30 patients; 18 patients (60%) were serum HBsAg positive. In serum HBsAg-positive group, intrahepatic cccDNA was detected in 66.66% of non-cancerous tissues, but only in 5.55% of HCC tissue; intrahepatic cccDNA levels in non-cancerous tissues were significantly higher than those in HCC tissues (p < 0.001), and correlated with serum HBsAg (p < 0.01). Significant correlations between intrahepatic HBsAg and intrahepatic cccDNA were found in both non-cancerous tissues (p < 0.01) and HCC tissues (p < 0.05). We concluded that HBV cccDNA and intrahepatic HBsAg in HBV-associated HCC tissues were significantly reduced, as compared with matched non-cancerous tissues. This warrants further investigation into the impacts and the cause(s) of cccDNA reduction in HBV-associated HCC tissues, which might yield novel immune-related therapy for HBV-associated HCC.

Expression of the microRNA-143/145 cluster is decreased in hepatitis B virus-associated hepatocellular carcinoma and may serve as a biomarker for tumorigenesis in patients with chronic hepatitis B.

The aims of the present study were to identify the expression profile of microRNA (miR)-143/145 in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC), explore its association with prognosis and investigate whether the serum miR-143/145 expression levels may serve as a diagnostic indicator of HBV-associated HCC. The microRNA (miRNA) chromatin immunoprecipitation dataset was obtained from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus databases, and analyzed using the Wilcoxon signed-rank test. It was observed that the expression of miR-143 and miR-145 was decreased 1.5-fold in HBV-associated HCC samples compared with non-tumor tissue in the TCGA and the GSE22058 datasets (P<0.01). Using the reverse transcription-quantitative polymerase chain reaction, it was further confirmed that miR-143/145 and their host gene MIR143HG were downregulated in HBV-associated HCC tissues compared with corresponding distal non-tumor tissues. The lower level of miR-143 and miR-145 expression was associated with tumor differentiation, and may thus be responsible for a poor prognosis of patients with HBV-associated HCC. The receiver-operating characteristic (ROC) curves were used to explore the potential value of miR-143 and miR-145 as biomarkers for predicting HBV-associated HCC tumorigenesis. In serum, miR-143/145 were identified to be significantly decreased in patients with HBV-associated HCC compared with negative control patients, and their associated areas under the ROC curves were calculated at 0.813 and 0.852 (P<0.05), with each having a sensitivity and a specificity close to 0.80. These results indicated that the decreased expression of the miR-143/145 cluster and their host gene MIR143HG in HBV-associated HCC tissue was associated with prognosis, and each of these miRNAs may serve as a valuable diagnostic biomarker for predicting HBV-associated HCC tumorigenesis.

Tripartite Motif Containing 52 (TRIM52) Promotes Cell Proliferation in Hepatitis B Virus-Associated Hepatocellular Carcinoma.

BackgroundChronic hepatitis B virus (HBV) infection is the major cause of hepatocellular carcinoma (HCC). HBV X protein (HBx) plays a crucial role in the development of HCC. Moreover, many tripartite motif (TRIM) family proteins exert diverse biological functions in hepatocarcinogenesis. However, as a novel member of this family, the specific effect of TRIM52 is still largely obscure. In the present study, we investigated the expression and function of TRIM52 in HBV-associated HCC.Material/MethodsFluorescence quantitative polymerase chain reaction (FQ-PCR) was performed to detect the HBV DNA levels in the peripheral blood of HCC patients. Quantitative real-time PCR (qRT-PCR) and Western blot analysis were performed to detect the expression of TRIM52, HBx, and NF-κB p65. HBx-pcDNA3.1 and TRIM52-shRNA were used to induce HBx ectopic expression and TRIM52 silencing, respectively. Pyrrolidine dithiocarbamate (PDTC) was used to block the activation of NF-κB. Cell proliferation was detected using the Cell Counting Kit-8 (CCK-8) assay.ResultsTRIM52 expression was up-regulated together with HBx in HBV-associated HCC tissues. Ectopic expression of HBx elevated TRIM52 expression in HepG2 cells. TRIM52 silencing repressed the proliferation of HepG2.2.15 cells. Moreover, NF-κB p65 expression was increased in HCC cell lines. Blocking NF-κB activation with PDTC suppressed TRIM52 expression and attenuated the viability of HepG2.2.15 cells.ConclusionsThese findings indicate that TRIM52 can promote cell proliferation and HBx may regulate TRIM52 expression via the NF-κB signaling pathway in HBV-associated HCC.

Hepatitis B virus X protein promotes hepatocellular carcinoma invasion and metastasis via upregulating thioredoxin interacting protein.

Hepatitis B virus X protein (HBx), a multifunctional protein encoded by the X gene of the hepatitis B virus (HBV) is involved in the metastasis of HBV-associated hepatocellular carcinoma (HCC) through various pathways, including upregulating intracellular reactive oxygen species (ROS). Thioredoxin interacting protein (TXNIP) is a key mediator of intracellular ROS, but its function in HBx-mediated metastasis of HBV-associated HCC is elusive. In the present study, HBV-associated HCC tissues with or without metastasis and HepG2 cells were used to study the function of TXNIP in HBx-mediated metastasis of HBV-associated HCC. Initially, the expression levels of TXNIP and HBx in HBV-associated HCC tissues were detected by immunohistochemistry and reverse transcription-quantitative polymerase chain reaction. The results revealed that high expression of TXNIP may be an independent risk factor for metastasis of HBV-associated HCC, and the mRNA levels of TXNIP and HBx were positively associated. Secondly, the association between HBx and TXNIP was investigated using a HBx expression stable cell line, in which HBx expression was induced and controlled by doxycycline. The results demonstrated that HBx may upregulate TXNIP expression in HepG2 cells. Thirdly, the effects of TXNIP and HBx on HepG2 cell migration and invasion were studied by scratch and Matrigel invasion assays, respectively. The results demonstrated that TXNIP overexpression enhanced HepG2 cell migration and invasion. In addition, ectopic expression of HBx promoted HepG2 cell migration and invasion, and this effect may be attenuated by knockdown of TXNIP expression, which indicated that TXNIP may be involved in the process. In summary, the present results demonstrated that TXNIP may be involved in HBx-mediated metastasis of HBV-associated HCC.

Hepatitis B virus X protein influences enrichment profiles of H3K9me3 on promoter regions in human hepatoma cell lines.

We previously showed that hepatitis B virus (HBV) X protein (HBx) could promote the trimethylation of histone H3 lysine 9 (H3K9me3) to repress tumor suppressor genes in hepatocellular carcinoma (HCC). In this work, we analyze 23,148 human promoters using ChIP-chip to determine the effects of HBx on H3K9me3 enrichments in hepatoma cells with transfection of HBx-expressing plasmid. Immunohistochemistry for HBx and H3K9me3 was performed in 21 cases of HBV-associated HCC tissues. We identified that H3K9me3 immunoreactivity was significantly correlated with HBx staining in HCC tissues. ChIP-chip data indicated that HBx remarkably altered promoter enrichments of H3K9me3 in hepatoma cells. We identified 25 gene promoters, whose H3K9me3 enrichments are significantly altered in hepatoma cells transfected HBx-expressing plasmid, including 19 gaining H3K9m3, and six losing this mark. Most of these genes have not been previously reported in HCC, and BTBD17, MIR6089, ZNF205-AS1 and ZP1 have not previously been linked to cancer; only two genes (DAB2IP and ZNF185) have been reported in HCC. Genomic analyses suggested that genes with the differential H3K9me3 enrichments function in diverse cellular pathways and many are involved in cancer development and progression.

MiR-106b promotes cancer progression in hepatitis B virus-associated hepatocellular carcinoma.

To investigate the effect of miR-106b on tumor progression in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC). A total of 120 patients who underwent liver resection for HCC at National Cheng Kung University Hospital were enrolled in the present study. MicroRNA (miRNA) array was first used to screen the miRNA expression profiles in HCC patients. The clinical records were retrospectively analyzed, and correlations with the miRNA expression profiles were evaluated. The mRNA expression levels of the miR-106b-25 cluster (miR-106b, miR-93 and miR-25), and MCM7 in tumor and non-tumor samples were quantitated using quantitative real-time reverse transcription-polymerase chain reaction (q-RT-PCR) analysis, and correlations in the levels of miR-106b, miR-93 and miR-25 expression were calculated. Kaplan-Meier overall and disease-free survival rates of HBV-associated HCC patients were analyzed using the log-rank test based on miR-106b expression. The comparison of the miR-106b expression levels in patients with different clinical outcomes was analyzed using Mann-Whitney U tests. Furthermore, a hepatitis B virus X protein (HBx) expression plasmid was transfected into Huh7 and Hep 3B cells. The expression levels of the miR-106b-25 cluster and MCM7 in HBx-expressing Huh7 and Hep 3B cells were detected using q-RT-PCR. miRNA array screening showed that miR-106b and its cluster, miR-93 and miR-25 were up-regulated in HCC patients (P < 0.01). The value of miR-106b expression in HBV-associated HCC patients was significantly higher than that in HCV- (P < 0.05) or non-B/non-C- (P < 0.001) associated HCC patients. The expression of the miR-106b-25 cluster was significantly higher in tumor tissue (P < 0.001) and associated with the host gene, MCM7, in clinical specimens from HBV-associated HCC patients. Furthermore, the expression levels of miR-106b, miR-93 and miR-25 were positively correlated in HBV-associated HCC tissues (miR-106 vs miR-93, r = 0.75; miR-93 vs miR-25, r = 0.69; miR-106b vs miR-25, r = 0.33). The overall and disease-free survival curves showed that high-miR-106b expression was correlated with the poor prognosis of HBV-associated HCC. HCC differentiation was significantly correlated with miR-106b expression (P < 0.05). Lower miR-106b expression levels resulted in the well differentiation of HCC. Moreover, the expression of the miR106b-25 cluster and MCM7 was up-regulated in Huh7 and Hep 3B cells after transfection with the HBx expression plasmid. The data obtained in the present study suggests that HBx enhances miR-106b transcription to promote tumor progression in HBV-associated HCC.

Hepatitis B virus X protein activates Notch signaling by its effects on Notch1 and Notch4 in human hepatocellular carcinoma.

Deregulated expression of Notch receptors and abnormal activity of Notch signaling have been observed in a growing number of malignant tumors, however, the expression and activity of Notch in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) and their relationship with HBV X protein (HBx) are still not fully elucidated. To address this, we examined the overall expression of Notch receptors in HBV-associated HCC tissues, analyzed their relationship with HBx, and further investigated the role of Notch signaling in HBx stable transfected HepG2 cells (HepG2X). The results showed that Notch signaling could be activated by HBx in HepG2 cells. The expression of cytoplasmic Notch1 or nuclear Notch4 was correlated with the expression of HBx in HBV-associated HCC tissues. The expression of cytoplasmic Notch1 or nuclear Notch4 could also be upregulated by HBx in HepG2X cells. The upregulation of Notch1 by HBx was through p38 MAPK pathway. Moreover, HBx was found to directly interact with Notch1, whereas, not with Notch4 in HepG2X cells. Suppression of Notch signaling by γ-secretase inhibitor (GSI) decreased cell growth, blocked cell cycle progression and induced cell apoptosis in HepG2X cells. The present study indicates that HBx activates Notch signaling by its effects on Notch1 and Notch4, and therefore, recruits Notch signaling as a downstream pathway contributing to its carcinogenic role in HBV-associated HCC.

Glucocorticoid-induced S-Adenosylmethionine Enhances the Interferon Signaling Pathway by Restoring STAT1 Protein Methylation in Hepatitis B Virus-infected Cells

Patients with chronic hepatitis B usually exhibit a low response to treatment with interferon α (IFN-α). An alternative approach to increase the response rate of IFN-α might be to immunologically stimulate the host with glucocorticoids (GCs) before treatment with IFN-α, but the underlying mechanism remains unclear. We hypothesized that the GCs enhance IFN signaling by inducing S-adenosylmethionine (AdoMet) when hepatitis B virus (HBV) replication was effectively suppressed by IFN-α. Here, we investigated the effect of GCs and IFN-α on AdoMet production and methionine adenosyltransferase 1A (MAT1A) expression in vitro. Furthermore, we determined whether post-transcriptional regulation is involved in HBV-repressed MAT1A expression and AdoMet production induced by dexamethasone (Dex). We found that AdoMet homeostasis was disrupted by Dex and that Dex directly regulated MAT1A expression by enhancing the binding of the glucocorticoid receptor (GR) to the glucocorticoid-response element (GRE) of the MAT1A promoter. HBV reduced AdoMet production by increasing methylation at GRE sites within the MAT1A promoter. The X protein of hepatitis B virus led to hypermethylation in the MAT1A promoter by recruiting DNA methyltransferase 1, and it inhibited GR binding to the GRE in the MAT1A promoter. Dex could increase an antiviral effect by inducing AdoMet production via a positive feedback loop when HBV is effectively suppressed by IFN-α, and the mechanism that involves Dex-induced AdoMet could increase STAT1 methylation rather than STAT1 phosphorylation. These findings provide a possible mechanism by which GC-induced AdoMet enhances the antiviral activity of IFN-α by restoring STAT1 methylation in HBV-infected cells.

HBx mutants differentially affect the activation of hypoxia-inducible factor-1α in hepatocellular carcinoma

Background:Mutations in HBx gene are frequently found in HBV-associated hepatocellular carcinoma (HCC). Activation of hypoxia-inducible factor-1α (HIF-1α) contributes to HCC development and progression. Wild-type HBx has been demonstrated to activate HIF-1α, but the effect of HBx mutations on HIF-1α has not been elucidated.Methods:HBx mutations were identified by gene sequencing in 101 HCC tissues. Representative HBx mutants were cloned and transfected into HCC cells. Expression and activation of HIF-1α were analysed by western blot and luciferase assays, respectively. The relationship between HBx mutants and HIF-1α expression in HCC tissues was also evaluated.Results:The dual mutations K130M/V131I enhanced the functionality of HBx as they upregulated the expression and transcriptional activity of HIF-1α. The C-terminal truncations and deletion mutations, however, weakened the ability of HBx to upregulate HIF-1α. Meanwhile, the C-terminus was further found to be essential for the stability and transactivation of HBx. In the HCC tissues, there was a positive association between the HBx mutants and HIF-1α expression.Conclusion:Different mutations of HBx exert differentiated effects on the functionality of HIF-1α, however, the overall activity of HBx mutants appears to increase the expression and transcriptional activity of HIF-1α.