HBV-negative Hepatocellular Carcinoma Research Articles

MiR-HCC2 suppresses hepatitis B virus replication by inhibiting the activity of the enhancer I/X promoter.

miR-HCC2 has been reported to markedly promote the growth, metastasis, and stemness of hepatocellular carcinoma (HCC) cells in vitro and in vivo. Deep sequencing showed that miR-HCC2 was significantly upregulated in hepatitis B virus (HBV)-positive (HBV+) HCC tissue samples compared with HBV-negative (HBV-) HCC tissue samples. miR-HCC2 expression was further evaluated in HCC tissues and cells, and the expression of miR-HCC2 was found to be significantly higher in HBV+ HCC tissues and cells than in HBV- HCC tissues and cells, suggesting that high miR-HCC2 expression could be induced by HBV infection. To explore the relationship between miR-HCC2 and HBV, we investigated the effect of miR-HCC2 on HBV antigen expression, transcription, and replication. We found that miR-HCC2 was involved in the negative feedback regulation of HBV replication. Further mechanistic studies revealed that miR-HCC2 suppressed HBV replication by inhibiting the activity of the enhancer I/Xpromoter. Our study demonstrates the effect of the inhibition of miR-HCC2 on HBV gene expression and replication, which can help to illustrate the complex regulatory network involving host miRNAs and HBV.

Ferroptosis-Related Gene SLC1A5 Is a Novel Prognostic Biomarker and Correlates with Immune Microenvironment in HBV-Related HCC

(1) Background: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide with limited treatment satisfaction. Finding new therapeutic targets has remained a major challenge. Ferroptosis is an iron-dependent cell death program that plays a regulatory role in HBV infection and HCC development. It is necessary to classify the roles of ferroptosis or ferroptosis-related genes (FRGs) in HBV-related HCC progression. (2) Methods: We conducted a matched case-control study from the TCGA database, retrospectively collecting demographic data and common clinical indicators from all subjects. The Kaplan-Meier curve, univariate and multivariate cox regression analysis of the FRGs were used to explore the risk factors for HBV-related HCC. The CIBERSORT algorithm and TIDE algorithm were executed to evaluate the functions of FRGs in the tumor-immune environment. (3) Results: A total of 145 HBV-positive HCC patients and 266 HBV-negative HCC patients were enrolled in our study. Four ferroptosis related genes (FANCD2, CS, CISD1 and SLC1A5) were positively correlated with the progression of HBV-related HCC. Among them, SLC1A5 was an independent risk factor for HBV-related HCC, and correlated with poor prognosis, advanced progression and an immunosuppression microenvironment. (4) Conclusions: Here, we revealed that a ferroptosis-related gene, SLC1A5, may be an excellent predictor of HBV-related HCC and may provide insight into the development of innovative possible therapeutic techniques.

The Intratumoral Bacterial Metataxonomic Signature of Hepatocellular Carcinoma.

ABSTRACTMicrobiota is implicated in hepatocellular carcinoma (HCC). The spectrum of intratumoral microbiota associated with HCC progression remains elusive. Fluorescence in situ hybridization revealed that microbial DNAs were distributed in the cytosol of liver hepatocytes and erythrocytes. Viable anaerobic or aerobic bacteria were recovered in HCC tissues by fresh tissue culture. We performed a comprehensive DNA sequencing of bacterial 16S rRNA genes in 156 samples from 28 normal liver, 64 peritumoral, and 64 HCC tissues, and the DNA sequencing yielded 4.2 million high-quality reads. Both alpha and beta diversity in peritumor and HCC microbiota were increased compared to normal controls. The most predominant phyla in HCC were Patescibacteria, Proteobacteria, Bacteroidota, Firmicutes, and Actinobacteriota. phyla of Proteobacteria, Firmicutes, and Actinobacteriota, and classes of Bacilli and Actinobacteria, were consistently enriched in peritumor and HCC tissues, while Gammaproteobacteria was especially abundant in HCC tissues compared to normal controls. Streptococcaceae and Lactococcus were the marker taxa of HCC cirrhosis. The Staphylococcus branch and Caulobacter branch were selectively enriched in HBV-negative HCCs. The abundance of Proteobacteria, Gammaproteobacteria, Firmicutes, Actinobacteriota, and Saccharimonadia were associated with the clinicopathological features of HCC patients. The inferred functions of different taxa were changed between the microbiota of normal liver and peritumor/HCC. Random forest machine learning achieved great discriminative performance in HCC prediction (area under the curve [AUC] = 1.00 in the training cohort, AUC = 0.950 for top five class signature, and AUC = 0.943 for the top 50 operational taxonomy units [OTUs] in the validation cohort). Our analysis highlights the complexity and diversity of the liver and HCC microbiota and established a specific intratumoral microbial signature for the potential prediction of HCC.IMPORTANCE Gut microbiome is an important regulator of hepatic inflammation, detoxification, and immunity, and contributes to the carcinogenesis of liver cancer. Intratumoral bacteria are supposed to be closer to the tumor cells, forming a microenvironment that may be relevant to the pathological process of hepatocellular carcinoma (HCC). However, the presence of viable intratumoral bacteria remains unclear. It is worth exploring whether the metataxonomic characteristics of intratumoral bacteria can be used as a potential marker for HCC prediction. Here, we present the first evidence of the existence of viable intratumoral bacteria in HCC using the tissue culture method. We revealed that microbial DNAs were distributed in the cytosol of liver hepatocytes and erythrocytes. We analyzed the diversity, structure, and abundance of normal liver and HCC microbiota. We built a machine learning model for HCC prediction using intratumoral bacterial features. We show that specific taxa represent potential targets for both therapeutic and diagnostic interventions.

Hepatitis B Virus Promotes Hepatocellular Carcinoma Progression Synergistically With Hepatic Stellate Cells via Facilitating the Expression and Secretion of ENPP2.

Background: Hepatitis B virus (HBV) infection is a major risk factor causing hepatocellular carcinoma (HCC) development, but the molecular mechanisms are not fully elucidated. It has been reported that virus infection induces ectonucleotide pyrophosphatase-phosphodiesterase 2 (ENPP2) expression, the latter participates in tumor progression. Therefore, the aim of the present study was to investigate whether HBV induced HCC malignancy via ENPP2. Methods: HCC patient clinical data were collected and prognosis was analyzed. Transient transfection and stable ectopic expression of the HBV genome were established in hepatoma cell lines. Immunohistochemical staining, RT-qPCR, western blot, and ELISA assays were used to detect the expression and secretion of ENPP2. Finally, CCK-8, colony formation, and migration assays as well as a subcutaneous xenograft mouse model were used to investigate the influence of HBV infection, ENPP2 expression, and activated hepatic stellate cells (aHSCs) on HCC progression in vitro and in vivo. Results: The data from cancer databases indicated that the level of ENPP2 was significant higher in HCC compared within normal liver tissues. Clinical relevance analysis using 158 HCC patients displayed that ENPP2 expression was positively correlated with poor overall survival and disease-free survival. Statistical analysis revealed that compared to HBV-negative HCC tissues, HBV-positive tissues expressed a higher level of ENPP2. In vitro, HBV upregulated ENPP2 expression and secretion in hepatoma cells and promoted hepatoma cell proliferation, colony formation, and migration via enhancement of ENPP2; downregulation of ENPP2 expression or inhibition of its function suppressed HCC progression. In addition, aHSCs strengthened hepatoma cell proliferation, migration in vitro, and promoted tumorigenesis synergistically with HBV in vivo; a loss-function assay further verified that ENPP2 is essential for HBV/aHSC-induced HCC progression. Conclusion: HBV enhanced the expression and secretion of ENPP2 in hepatoma cells, combined with aHSCs to promote HCC progression via ENPP2.

Comprehensive genomic profiling analysis of HBV-infected hepatocellular carcinoma patients.

e16180 Background: Hepatocellular carcinoma (HCC) is one of the leading causes of tumor-related death worldwide which is dominantly caused by chronic infection of hepatitis B virus (HBV) and hepatitis C virus (HCV). Recent studies have shown that immune checkpoint inhibitors may enter the first-line treatment of HCC. Comprehensive investigation of the genetic alterations and their correlation with microsatellite instability (MSI) and PD-L1 expression in HBV-infected HCC is helpful to determine the therapy strategy for patients. Methods: The genomic information of HCC patients was obtained from HapLab database. HaploX 605- gene panel was performed to analyze the genomic data of patients. PD-L1 expression was detected by immunochemistry. Results: A total of 349 mutations were found by analyzing the genomic profiles of 115 HBV-infected HCC patients. The frequently mutated genes included TP53 (63%), TERT (27%), CTNNB1 (16%), CSMD3 (15%), LRP1B (14%), AXIN1 (13%), CDKN2A (13%), KIT (13%), MDM4 (12%) and CCND1 (12%). Besides, 279 mutations were identified by analyzing the genomic profiles of 82 HCC patients without HBV-infection. Mutation frequencies for several genes were relatively higher in the HBV-positive patients than that in HBV-negative HCC patients, including TP53 (63% versus 39%), CCND1 (12% versus 5%), RB1(12% versus 4%) and BRCA2 (11% versus 4%). The proportion of MSI-H/L in HBV-infection patients were higher than that in HBV-negative ones (7.83% versus 4.88%). Notably, the proportion of high PD-L1 expression in patients with HBV-infection was 40.57% (43/106), while it was 28.95% (22/76) in patients without HBV-infection. Conclusions: These results highlighted a potential impact of viral infection on the mutational signatures in hepatocarcinogenesis. The PD-L1 expression status and MSI were closely related to HBV-infected HCC. These results may contribute to understanding the mechanism of HBV-infected HCC and the selection of therapy for relative patients.

Hepatitis B virus P protein initiates glycolytic bypass in HBV-related hepatocellular carcinoma via a FOXO3/miRNA-30b-5p/MINPP1 axis

BackgroundHepatitis B virus (HBV) infection is a crucial risk factor for hepatocellular carcinoma (HCC). However, its underlying mechanism remains understudied.MethodsMicroarray analysis was conducted to compare the genes and miRNAs in liver tissue from HBV-positive and HBV-negative HCC patients. Biological functions of these biomarkers in HBV-related HCC were validated via in vitro and in vivo experiments. Furthermore, we investigated the effect of HBV on the proliferation and migration of tumor cells in HBV-positive HCC tissue. Bioinformatics analysis was then performed to validate the clinical value of the biomarkers in a large HCC cohort.ResultsWe found that a gene, MINPP1 from the glycolytic bypass metabolic pathway, has an important biological function in the development of HBV-positive HCC. MINPP1 is down-regulated in HBV-positive HCC and could inhibit the proliferation and migration of the tumor cells. Meanwhile, miRNA-30b-5p was found to be a stimulator for the proliferation of tumor cell through glycolytic bypass in HBV-positive HCC. More importantly, miRNA-30b-5p could significantly downregulate MINPP1 expression. Metabolic experiments showed that the miRNA-30b-5p/MINPP1 axis is able to accelerate the conversion of glucose to lactate and 2,3-bisphosphoglycerate (2,3-BPG). In the HBV-negative HCC cells, miRNA-30b-5p/MINPP1 could not regulate the glycolytic bypass to promote the tumorigenesis. However, once HBV was introduced into these cells, miRNA-30b-5p/MINPP1 significantly enhanced the proliferation, migration of tumor cells, and promoted the glycolytic bypass. We further revealed that HBV infection promoted the expression of miRNA-30b-5p through the interaction of HBV protein P (HBp) with FOXO3. Bioinformatics analysis on a large cohort dataset showed that high expression of MINPP1 was associated with favorable survival of HBV-positive HCC patients, which could lead to a slower progress of this disease.ConclusionOur study found that the HBp/FOXO3/miRNA-30b-5p/MINPP1 axis contributes to the development of HBV-positive HCC cells through the glycolytic bypass. We also presented miRNA-30b-5p/MINPP1 as a novel biomarker for HBV-positive HCC early diagnosis and a potential pharmaceutical target for antitumor therapy.

IQGAP1 promotes anoikis resistance and metastasis through Rac1-dependent ROS accumulation and activation of Src/FAK signalling in hepatocellular carcinoma.

BackgroundHepatitis B virus (HBV) has a crucial role in the progression of hepatocellular carcinoma (HCC). Tumour cells must develop anoikis resistance in order to survive before metastasis. This study aimed to investigate the mechanism of IQGAP1 in HBV-mediated anoikis evasion and metastasis in HCC cells.MethodsIQGAP1 expression was detected by immunohistochemistry, real-time PCR and immunoblot analysis. Lentiviral-mediated stable upregulation or knockdown of IGAQP1, immunoprecipitation, etc. were used in function and mechanism study.ResultsIQGAP1 was markedly upregulated in HBV-positive compared with HBV-negative HCC cells and tissues. IQGAP1 was positively correlated to poor prognosis of HBV-associated HCC patients. IQGAP1 overexpression significantly enhanced the anchorage-independent growth and metastasis, whereas IQGAP1-deficient HCC cells are more sensitive to anoikis. Mechanistically, we found that HBV-induced ROS enhanced the association of IQGAP1 and Rac1 that activated Rac1, leading to phosphorylation of Src/FAK pathway. Antioxidants efficiently inhibited IQGAP1-mediated anoikis resistance and metastasis.ConclusionsOur study indicated an important mechanism by which upregulated IQGAP1 by HBV promoted anoikis resistance, migration and invasion of HCC cells through Rac1-dependent ROS accumulation and activation of Src/FAK signalling, suggesting IQGAP1 as a prognostic indicator and a novel therapeutic target in HCC patients with HBV infection.

Distinct clinical and genetic characteristics of AFP-negative hepatocellular carcinoma patients infected by HBV or HCV.

e16620 Background: The serum level of alpha-fetoprotein (AFP) is a key biomarker in hepatocellular carcinoma (HCC) screening regardless of the high false-negative rates. Studies have characterized AFP negative HCC patients, however, the differences between HBV and HCV infected AFP negative HCC patients have not been discussed. Methods: The clinical profiles and whole-exome sequencing data of 243 patients were downloaded from the TCGA database. Patients were divided into 4 groups based on their virus infection status and serum AFP measurements as indicated in the table. The mutation profiles and clinical features of the patients were analyzed. Results: In the HBV infected groups, the AFP+ patients carried significantly larger tumors (n = 17, 85.88±11.87 mm) compared to the AFP- patients (n = 13, 49.08±8.933 mm, p = 0.026). The HBV+ AFP+ group exhibited the prevalence of vascular invasion (VI, 76%, 13/17) while less than half of the patients in the HBV+ AFP- group developed VI (46%, 6/13). The WES results identified shared mutations in more than 10% of patients in each group. It appeared that 126 shared mutations were restricted to the HBV+ AFP+ group, they are involved in the regulation of ion transmembrane transport (p < 0.001) and interaction between L1 and Ankyrins (p < 0.0001). Only 51 shared mutations were exclusive to the HBV+ AFP- group, they majorly affect developmental growth (p < 0.001) and skeletal system development (p < 0.01). In contrast, the tumor sizes of HCV+ AFP+ and HCV+ AFP- patients are similar (55.18±7.781 vs 48.19±5.564, p = 0.47), while the VI rate is higher in the HCV+ AFP- group (63%, 17/29, Table). In total, 11 shared mutations were restricted to the HCV+ AFP+ group, several of the genes were involved in the generation of precursor metabolites and energy (p < 0.01) and activation of immune response (p < 0.01). 27 shared mutations were exclusive to the HCV+ AFP- group, the enrichment analysis showed that they are related to the regulation of synapse organization (p < 0.0001) and the regulation of chromosome organization (p < 0.01). Conclusions: HBV and HCV infection were associated with distinct clinical features among the AFP positive and AFP negative patients. The differences were underpinned by the mutations exclusive to each group, and they were involved in distinct molecular and cellular functions. [Table: see text]

A novel miR-0308-3p revealed by miRNA-seq of HBV-positive hepatocellular carcinoma suppresses cell proliferation and promotes G1/S arrest by targeting double CDK6/Cyclin D1 genes

BackgroundPersistent infection with hepatitis B virus (HBV) accounts for the majority of hepatocellular carcinoma (HCC), but the molecular mechanisms underlying liver carcinogenesis are still not completely understood. Increasing evidence demonstrates that microRNAs (miRNAs) play significant functional roles in virus–host interactions. The aim of this study was to explore differentially expressed miRNA profiles and investigate the molecular mechanism of miR-0308-3p in HBV-positive HCC carcinogenesis.MethodsHigh-throughput sequencing was used to detect novel miRNAs in three samples of HBV-positive HCC tissue compared to matched HBV-negative HCC tissue. The Cancer Genome Atlas (TCGA) database was used to mine miRNAs related to HBV-positive HCC. Bioinformatics analyses were conducted to predict the miRNAs’ possible biological and pathway regulatory functions. Quantitative polymerase chain reaction (qPCR) was then applied to evaluate the expression levels of randomly selected miRNAs. CCK-8 was used to measure cell proliferation and cell cycles were analyzed using flow cytometry. A dual luciferase reporter gene assay was used to confirm the downstream targets of miR-0308-3p.ResultsIn total, there were 34 overlapping miRNAs in both our miRNA-seq data and the TCGA database. We found two overlapping miRNAs in both the HBV-positive HCC samples and the TCGA database, and 205 novel pre-miRNA sequences were predicted. miR-522 and miR-523 were markedly overexpressed in HBV-positive HCC and were associated with a significantly poorer long-term prognosis (miR-522, HR 2.19, 95% CI 1.33–3.6, p = 0.0015; miR-523HR 1.5, 95% CI 1–2.44, p = 0.0047). Of note, we found that the novel miR-0308-3p was markedly downregulated in HBV-positive HCC samples and HCC cancer cell lines compared with HBV-negative HCC samples and adjacent normal hepatic tissue. Moreover, elevated expression of miR-0308-3p was found to inhibit proliferation of cancer cells by promoting G1/S cell cycle arrest but did not influence the apoptosis of cancer cells. A dual luciferase reporter activity assay identified that miR-0308-3p acted directly on the target sequence of the CDK6 and Cyclin D1 mRNA 3ʹUTR to suppress CDK6 and Cyclin D1 expression.ConclusionsMiR-0308-3p upregulation dramatically suppressed HCC cell proliferation and induced G1/S cell cycle arrest by directly targeting CDK6/Cyclin D1. These findings reveal a novel molecular mechanism for activation of G1/S arrest in HCC and may prove clinically useful for developing new therapeutic targets.

HBx-induced S100A9 in NF-\u03baB dependent manner promotes growth and metastasis of hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is associated with hepatitis B virus (HBV) infection. Myeloid-specific S100 proteins (S100s), namely, S100A8, S100A9 and S100A12, have been recently recognized as newly discovered damage-associated molecular patterns (DAMPs) that are correlated with progression in pathogen of infectious diseases. However, whether S100s are regulated by HBV and involved in HBV-related hepatocarcinogenesis are still unclear. Here, we found that all expression levels of myeloid-specific S100s (S100A8, S100A9 and S10012) were elevated in serum and tissue samples from HCC patients. Expression of S100A9 but not S100A8 and S10012 were also higher in blood serum and tissue samples from HBV-positive HCC patients than that in HBV-negative HCC patients. High levels of intracellular and extracellular S100A9 were also confirmed in HepG2 cells expressing 1.3-fold HBV genome or HBV-encoded X protein (HBx) as well as in a stable HBV-producing cell line HepG2.2.15. HBx was shown to facilitate translocation of NF-κB from the cytoplasm to the nucleus, and NF-κB bound to the promoter of S100A9 to enhance its transcription. Silencing S100A9 expression partially blocked HBx-induced growth and metastasis of HepG2 cells both in vitro and in vivo. Further, serum S100A9 levels were found to correlate with TNM stage, extrahepatic metastasis status and HBV DNA load in HBV-related HCC and also had a better diagnostic value for identifying extrahepatic metastasis. Our these data demonstrate that S100A9 plays a pivotal role in HBx-induced growth and metastasis of HCC and may serve as a potential diagnostic marker for extrahepatic metastasis.

The expression and role of lncRNA AX800134 in hepatitis B virus-related hepatocellular carcinoma.

Chronic infection with hepatitis B virus (HBV) is one of most important risk factors for the development of hepatocellular carcinoma (HCC). Several long non-coding RNAs (lncRNAs) have been shown to be involved in the etiology of HBV-related HCC. AX800134 is one recently identified lncRNA associated with HCC. In this study, we validated the upregulated expression of AX800134 in HBV-positive HCC compared with HBV-negative HCC. Furthermore, we found that HBV X protein (HBx) directly triggered AX800134 expression in human hepatoma HepG2 cells. Pro-inflammatory cytokine TNFα also induced AX800134 upregulation in HBx-expressing HepG2 cells, which could be reversed by reactive oxygen species (ROS) scavenger pyrrolidine dithiocarbamate (PDTC). Additionally, silencing AX800134 with siRNA interference remarkably inhibited the growth and invasion of HBx-expressing HepG2 cells. AX800134 antagonism also enhanced spontaneous apoptosis of HepG2 cells under serum deprivation condition. Therefore, our results indicate that highly expressed AX800134 acts as an oncogenic factor in HCC, and its upregulation is related with the viral product HBx and chronic inflammation.

Hepatitis transactivator protein X promotes extracellular matrix modification through HIF/LOX pathway in liver cancer

Hepatocellular carcinoma (HCC), accounting for 90% of primary liver cancer, is a lethal malignancy that is tightly associated with chronic hepatitis B virus (HBV) infection. HBV encodes a viral onco-protein, transactivator protein X (HBx), which interacts with proteins of hepatocytes to promote oncogenesis. Our current study focused on the interaction of HBx with a transcription factor, hypoxia-inducible factor-1α (HIF-1α), which is stabilized by low O2 condition (hypoxia) and is found to be frequently overexpressed in HCC intra-tumorally due to poor blood perfusion. Here, we showed that overexpression of HBx by tetracycline-inducible systems further stabilized HIF-1α under hypoxia in HBV-negative HCC cell lines. Reversely, knockdown of HBx reduced HIF-1α protein stabilization under hypoxia in HBV-positive HCC cell lines. More intriguingly, overexpression of HBx elevated the mRNA and protein expression of a family of HIF-1α target genes, the lysyl oxidase (LOX) family in HCC. The LOX family members function to cross-link collagen in the extracellular matrix (ECM) to promote cancer progression and metastasis. By analyzing the collagens under scanning electron microscope, we found that collagen fibers were significantly smaller in size when incubated with conditioned medium from HBx knockdown HCC cells as compared to control HCC cells in vitro. Transwell invasion assay further revealed that less cells were able to invade through the matrigel which was pre-treated with conditioned medium from HBx knockdown HCC cells as compared to control HCC cells. Orthotopic and subcutaneous HCC models further showed that knockdown of HBx in HCC cells reduced collagen crosslinking and stiffness in vivo and repressed HCC growth and metastasis. Taken together, our in vitro and in vivo studies showed the HBx remodeled the ECM through HIF-1α/LOX pathway to promote HCC metastasis.

MicroRNA profile in HBV-induced infection and hepatocellular carcinoma

BackgroundMicroRNAs (miRNAs) exhibit essential regulatory functions related to cell growth, apoptosis, development and differentiation. Dysregulated expression of miRNAs is associated with a wide variety of human diseases. As such miRNA signatures are valuable as biomarkers for disease and for making treatment decisions. Hepatitis B virus (HBV) is a major risk factor for hepatocellular carcinoma (HCC). Here we screened for miRNAs in chronic HBV associated HCC.MethodsTo determine the miRNAs in HCC occurrence associated with HBV infection, we analyzed global miRNA expression profiles in 12 pairs of HCC and adjacent matched non-HCC tissues from HBV-positive and HBV-negative patients using microarray analyses. The microarray result was validated by real-time PCR in 32 HBV-positive and 24 HBV-negative patient HCC samples. The potential candidate target genes of the miRNAs were predicted by miRWalk software. Genes simultaneously predicted as targets by two or more miRNAs were subjected to GO and KEGG pathway analysis. The miRNA regulatory network analysis was performed using the Ingenuity Pathway Analysis (IPA) software.ResultsEight miRNAs (miR-223, miR-98, miR-15b, miR-199a-5p, miR-19b, miR-22, miR-451, and miR-101) were involved in HBV-unrelated HCC, 5 miRNAs (miR-98, miR-375, miR-335, miR-199a-5p, and miR-22) were involved in HBV infection, and 7 miRNAs (miR-150, miR-342-3p, miR-663, miR-20b, miR-92a-3p, miR-376c-3p and miR-92b) were specifically altered in HBV-related HCC. Gene Ontology and KEGG analyses predict that these HBV-related HCC miRNAs are involved in the regulation of: transcription, RNA polymerase II promoter, phosphorylation of proteins through MAPK signaling pathway, focal adhesion, and actin cytoskeleton. IPA analysis also suggest that these miRNAs act on AGO2, TP53, CCND1, and 11 other genes that significantly influence HCC occurrence and HBV infection.ConclusionOur data indicates that the unique 7 miRNAs expression signature could be involved in the development HBV- related HCC.

Up-regulation of brain-expressed X-linked 2 is critical for hepatitis B virus X protein-induced hepatocellular carcinoma development.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Chronic hepatitis B virus (HBV) infection is a major cause for HCC. Hepatitis B virus X (HBx), one of four proteins encoded by HBV genome, plays a vital role in the pathogenesis of HBV-induced HCC. However, the molecular mechanisms of HBx-triggered HCC remain largely undetermined. Here we revealed that the expression of Brain-expressed X-linked 2 (BEX2) and Osteopontin (OPN) were elevated in liver tissues of HBV transgenic mice and human HCC specimens. Moreover, a positive correlation between BEX2 and OPN was exhibited in samples from HCC patients with HBV infection. The protein levels of BEX2 and OPN were both higher in HBV-positive HCC specimens compared to that of HBV-negative HCC specimens. HBx potentiated OPN expression through up-regulation of BEX2. Importantly, the depletion of BEX2 suppressed tumorigenic potential of HCC cells with highly expressed HBx. We demonstrated the important role of BEX2 in HCC pathogenesis, and BEX2 may be a novel therapeutic target for HCC patients with HBV infection. The newly identified HBx/BEX2/OPN signaling cassette is implicated in the pathogenesis of HBV-induced HCC.

Correlation of IP-10 gene expression with Serum alanine transaminase (ALT) levels and Hepatitis B viral load in cirrhosis and hepatocellular carcinoma (HCC) patients.

Interferon-gamma induced protein 10 (IP-10), a chemokine is suggested to be involved in liver injury during Hepatitis B virus infection (HBV). The increase of IP-10 is a critical step for recruitment of inflammatory cells to the local focus of the liver and hepatopathology. This study was designed to assess the correlation of IP-10 gene expression with HBV-DNA and serumAlanine transaminase(ALT) in patients with cirrhosis and HCC.The study was conducted among 60 patients. The study populationwere divided into four groups (15 in each groups)-HBV positive cirrhosis, HBV negative cirrhosis, HBV positiveHCC and HBV negative HCC. Expression of IP-10 gene was observed using real time PCR.IP-10 gene expressions in the above mentioned groups were correlated with serum ALT level and HBV viral load.IP-10 gene was significantly higher in HBV-positive patients with HCC than HBVpositive cirrhosis. Similarly, the expression of IP-10 was significantly higher in HBV-positive HCC than HBVnegative HCC patients. However, the expression of IP-10 was reduced in HBV-positive cirrhosis in comparison with HBV-negative cirrhosis.IP-10 gene expression in liver was not correlated with the serum levels of ALT in any of the study groups. HBV- DNA load also did not correlated with IP-10 gene expression in HBV positive HCC and HBV positive cirrhosis patients.This study shows that there was no significant change with the expression of IP-10 gene in any of the study groups with ALT level or viral load, though differential expression of IP-10 gene were observed in cirrhosis and HCC patients.
 Bangladesh J Med Microbiol 2016; 10 (2): 9-13

MiR-19a, miR-122 and miR-223 are differentially regulated by hepatitis B virus X protein and involve in cell proliferation in hepatoma cells.

BackgroundHepatitis B virus (HBV) X protein (HBx) is a type of oncogenic protein involved in the progression of hepatocellular carcinoma (HCC) via interacting with host genes. Dysregulation of microRNAs (miRNAs) has been observed in HCC. This study aimed to investigate the role of HBx protein in the regulation of miR-19a, miR-122 and miR-223, and examine if these miRNAs involve in progression of malignant hepatocytes.MethodsQuantitative real time PCR (qRT-PCR) was used to measure the expression of miR-19a, miR-122 and miR-223 in patient samples and in HepG2 cells transfected with HBx or 1.3 fold HBV genome and also in HepG2.2.15 cells, which stably produces HBV. Their target mRNAs and proteins-PTEN, cyclin G1 and c-myc were measured by qRT-PCR and western blot, respectively. The effect of miR-19a, miR-122 and miR-223, and their respective target genes, on cell proliferation was analyzed using 5-ethynyl-2-deoxyuridine incorporation and MTT assay.ResultsMiR-19a showed an up-regulation in HBV-positive HCC patients compared to healthy controls and HBV-negative HCC patients, while miR-122 and miR-223 showed a down-regulation compared to healthy controls, and miR-122 in HBV-positive HCC patients was also down-regulated when compared to HBV-negative HCC patients. MiR-19a was found to be up-regulated in HepG2 cells transfected with HBx or 1.3 fold HBV genome, but down-regulated in HepG2.2.15 cells. MiR-122 and miR-223 were down-regulated in HBx or 1.3 fold HBV transfected HepG2 cells as well as in HepG2.2.15 cell. Their target mRNAs and corresponding proteins-PTEN was down-regulated, while cyclin G1 and c-myc were found to be up-regulated. Modulated expression of miR-19a, miR-122 and miR-223 enhanced cell proliferation of HBx-transfected HepG2 cells, and rescue experiment further showed that their target genes-PTEN, cyclin G1and c-myc involved in cell proliferation of HBx-transfected HepG2 cells.ConclusionsThe expression of miR-19a, miR-122 and miR-223 were differentially regulated by HBx protein, the differential expression of miR-19a, miR-122 and miR-223 plays an important role in cell proliferation of HCC. This study provides new insight into understanding how HBx protein interacts with miRNAs and subsequently regulates host function.

Downregulation of MicroRNA-145 Caused by Hepatitis B Virus X Protein Promotes Expression of CUL5 and Contributes to Pathogenesis of Hepatitis B Virus-Associated Hepatocellular Carcinoma

Background: Hepatitis B viral infection-induced hepatocellular carcinoma (HCC) is a major threat to human health in China. Hepatitis B virus X protein (HBX), an HBV protein, has been reported to be involved in regulating the cellular activities of the host cells and is responsible for HCC oncogenesis. Methods and Results: In this study, we performed real-time PCR in tumor tissue samples collected from 53 HCC patients (25 HBV-positive cases and 28 HBV-negative cases) to screen the candidate miRNAs that have previously been reported to be aberrantly expressed in HBV-associated HCC and found that miR-145 was significantly downregulated. The following computational analysis identified CUL5 and RAB5C as virtual targets of miR-145, whereas only CUL5 was verified as a validated target gene of miR-145 in liver cells via luciferase reporter assay. In line with this result, we found that both the mRNA and protein expression levels of CUL5 were significantly higher in HBV-positive than in HBV-negative HCC. An in vitro experiment demonstrated a significant decrease in the expression of miRNA-145, a substantial increase in the mRNA and protein expression of CUL5, and an enhanced proliferation of HBX over-expressing HepG2 cells compared with the control. In HepG2.2.15, we found significant decreases in both the expression of CUL5 and the cell growth rate of H cells transfected with 60 nM miR-145 mimics compared with the scramble controls. Conclusion: HBV infection promotes cell growth, at least partially, through the HBX-induced downregulation of miRNA-145 expression, which is responsible for the oncogenesis of HBV-associated HCC.

HBV preS2 transactivates FOXP3 expression in malignant hepatocytes.

Recent data reported the increased expression of forkhead box protein 3 (FOXP3), the well known master regulator of CD4(+) C25(+) regulatory T cells, in hepatocellular carcinoma (HCC) cells. However, the mechanisms remain unknown. We previously showed that preS2, one of important regulatory proteins encoded by HBV, triggers transactivation of hTERT in malignant hepatocytes. Here, we aimed to explore the role of preS2 in regulating FOXP3 expression in HCC. FOXP3 expression was detected by RT-PCR, Western blot and immunohistochemical staining. Cotransfection and siRNA knockdown were involved to study the regulation effects of preS2 on FOXP3 expression in cultured HCC cell lines. Luciferase reporter assay and EMSA assay were performed to explore the mechanism of preS2-mediated FOXP3 upregulation. Immunohistochemical staining detected significant increased FOXP3 expression in malignant hepatocytes from sections of HCC patients. The total FOXP3 expression in hepatocytes from patients with HBsAg-positive HCC was significantly increased compared to that of HBV-negative HCCs (P = 0.002). In accordance, preS2 overexpression enhanced FOXP3 expression in HCC cell lines, while preS2 knockdown significantly reduced FOXP3 expression in HBV-integrated HepG2.2.15 cells. Results of cotransfection and luciferase report assay showed that preS2 transactivated FOXP3 promoter in a dose-dependent manner. Further study identified the AP-1 binding site at 20 bp region from -465 bp to -445 bp of FOXP3 promoter was responsible for preS2-induced FOXP3 transcriptional activation. Our data here, for the first time, provided direct evidence to demonstrate that preS2 oncoprotein encoded by HBV transactivated FOXP3 transcription in HCC cells.

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.

Correlation between androgen receptor expression and hepatitis B virus X protein and its clinical significance in hepatocellular carcinoma

To investigate the expression of androgen receptor (AR) and hepatitis B virus X protein (HBx) in hepatocellular carcinoma (HCC), and analyze the relationship between AR and HBx expressions. Tumor tissues and peritumoral tissues of 83 HBV-associated HCC cases were investigated in this study. Fourteen cases of HBV-negative HCC and 13 cases of hemangioma peritumoral tissues were considered as control. AR and HBx mRNA levels were determined by quantitative fluorescence real-time RT-PCR and their protein levels were assayed by Western blot. The expression of AR and HBx proteins in tissues were examined with EnVision immunohistochemical staining. The methylation status of AR promoter was determined using methylation-specific PCR (MSP). Both expression levels of AR mRNA and protein of the peritumoral tissues were significantly higher (0.17) than that of tumor tissues (0.09) in HBV-associated HCC (P < 0.01), but such a difference was not found in HBV-negative HCC (0.06 vs. 0.07, P > 0.05). The level of AR expression in peritumoral tissues was associated with tumor differentiation in HBV-associated HCC. AR mRNA and protein levels of peritumoral tissues in HBV-associated HCC were significantly higher than that in HBV-negative HCC and hemangioma (all P < 0.05). In the tumor tissues, HBV-associated HCC had significantly higher AR expression than HBV-negative HCC at mRNA level (P < 0.05), but not at protein level. Spearman rank correlation analysis showed that the AR mRNA or AR protein levels were positively correlated with HBx in both tumor and peritumoral tissues in HBV-associated HCC, but the expressions of AR and HBx were not associated with AR promoter methylation status. The relative expression levels of AR mRNA and protein in the HBV-associated peritumoral tissues were negatively correlated with tumor differentiation (r = -0.213, P < 0.05; r = -0.313, P < 0.05), the higher the AR expression, the poorer differentiation. But this correlation of AR mRNA and protein was not shown in the hepatocellular carcinoma tissues. HBx may enhance AR expression in HBV-associated HCC, but AR promoter demethylation maybe not been involved in its main mechanism. An increased AR expression is probably an early event during the development and progression of HBV-associated HCC, and AR expression in the peritumoral tissue is correlated with HBV-associated HCC differentiation. AR may play different roles in HBV-associated HCC and HBV-negative HCC.