Hepatitis B virus X protein-mediated upregulation of miR-221 activates the CXCL12-CXCR4 axis to promote NKT cells in HBVrelated hepatocellular carcinoma

Both hepatitis B virus X protein(HBx)and microRNA-221(miR-221)have been implicated in the development of hepatitis B virus(HBV)-related hepatocellular carcinoma(HCC).The present study demonstrates that HBx promotes HCC cell proliferation via the C-X-C motif chemokine ligand 12-C-X-C chemokine receptor type 4(CXCL12-CXCR4)axis.We predict that HBx/miR-221-mediated CXCL12/CXCR4 signaling induces NKT cells to promote HBV-related HCC.Methods:After miR-221 mimic,miR-221 mimic negative control,miR-221 inhibitor,miR-221 inhibitor negative control were transfected into cells,the expression of CXCL12 and miR-221 was detected by qPCR and western blot.Then we constructed a stable HBV-HCC cell line.HBV-HCC cells were injected into the nude mice,thus a HBV-HCC mouse model was constructed.Q-PCR and western blot were used to detect the expression of HBx,miR-221,CXCL12 and CXCR4 in tumor tissues.The expression of CXCL12 was detected by immunohistochemistry,and the expression of CXCR4,CD3 and CD56 was detected by immunofluorescence.The levels of CXCL12,IL-2 and TNF-αin serum of mice were detected by ELISA.Sixty-one patients with HBV-related HCC,61 patients with HBV-related cirrhosis,61 patients with chronic hepatitis B(CHB)and 30 healthy people were enrolled.CXCL12,cytokine levels,and clinicopathological parameters were tested.Results:Hepatitis B virus X protein upregulates the expression of miR-221 and CXCL12 in lentivirus(LV5)-HBx-transfected HepG2 cells.HBx protein promotes HepG2 cell proliferation in vitro.HBx protein promoted tumor growth via the miR-221/CXCL12/CXCR4 pathway in a mouse tumor model.HBx protein upregulated natural killer T cell expression via the CXCR4/CXCL12 pathway to promote tumor growth.The data demonstrated a positive correlation between CXCL12 concentration with Cre levels and Child-Pugh scores.CXCL12 had an inferior diagnostic efficiency compared to IL-2 and IL-6 for HBV-related HCC.Conclusions:We present evidence that HBx/miR-221-mediated CXCL12/CXCR4 signaling induces NKT cells to promote HBV-related HCC.

Hepatitis B virus X protein promotes liver cell proliferation via a positive cascade loop involving arachidonic acid metabolism and p-ERK1/2

Hepatitis B virus X protein （HBx） plays a crucial role in the development of hepatocellular carcinoma. Here, we sought to identify the mechanisms by which HBx mediates liver cell proliferation. We found that HBx upregulated the levels of cyclooxygenase-2 （COX-2）, 5-1ipoxygenase （5-LOX） and phosphorylated extracellular signal-regulated protein kinases 1/2 （p-ERK1/2） in liver cells. HBx-induced p-ERK1/2 was abolished by inhibition of Gi/o proteins, COX or LOX. In addition, HBx increased the amounts of prostaglandin E2 （PGE2） and leukotriene B4 （LTB4） released from cell lines derived from hepatocytes. Moreover, these released arachidonic acid metabolites were able to activate ERK1/2. Interestingly, activated ERK1/2 could upregulate the expression of COX-2 and 5-LOX in a positive feedback manner. In conclusion, HBx enhances and maintains liver cell proliferation via a positive feedback loop involving COX-2, 5-LOX, released arachidonic acid metabolites, Gi/o proteins and p-ERK1/2.

Hepatitis B virus X protein induces hepatic stem cell-like features in hepatocellular carcinoma by activating KDM5B

To determine the role of hepatitis B virus X protein (HBx), HBx in regulating hepatic progenitor cell (HPC)-like features in hepatocellular carcinoma (HCC) and the underlying molecular mechanisms.METHODSWe used a retrovirus vector to introduce wild type HBx or empty vector into HepG2 cells. We then used these cells to analyze cell proliferation, senescence, transformation, and stem-like features. Gene expression profiling was carried out on Affymetrix GeneChip Human U133A2.0 ver.2 arrays according to the manufacturer’s protocol. Unsupervised hierarchical clustering analysis and Class Comparison analysis were performed by BRB-Array Tools software Version 4.2.2. A total of 238 hepatitis B virus (HBV)-related HCC patients’ array data were used for analyzing clinical features.RESULTSThe histone demethylase KDM5B was significantly highly expressed in HBV-related HCC cases (P < 0.01). In HBV proteins, only HBx up-regulated KDM5B by activating c-myc. Hepatic stem cell (HpSC) markers (EpCAM, AFP, PROM1, and NANOG) were significantly highly expressed in KDM5B-high HCC cases (P < 0.01). KDM5B played an important role in maintaining HpSC-like features and was associated with a poor prognosis. Moreover, inhibition of KDM5B suppressed spheroid formation and cell invasion in vitro.CONCLUSIONHBx activates the histone demethylase KDM5B and induces HPC-like features in HCC. Histone demethylases KDM5B may be an important therapeutic target against HBV-related HCC cases.

The influence of hepatitis B virus X protein on the clock genes in liver cells and its significance

Objective： The aim of this study was to investigate the influence of hepatitis B virus X protein （HBx） on the clock genes in LO2 cells and its significance. Methods： A cell line LO2-HBx, Stably transfected with HBx gene, was established. The levels of mRNA and protein expression of CLOCK and BMAL1 were detected by real-time PCR and western blot. Resuits： The expression of CLOCK mRNA and protein were increased in cell line LO2-HBx （P 〈 0.05）, while the expression of BMAL1 mRNA and protein were decreased in cell line LO2-HBx （P 〈 0.05）. Conclusion： The expressions of core clock gene CLOCK and BMAL1 have been changed by HBx, which breaks down the previous circadian rhythm of liver cells. This maybe one of the reasons leads to the formation of liver cancer.

Interferon-alpha restrains growth and invasive potential of hepatocellular carcinoma induced by hepatitis B virus X protein

AIM: To investigate the effects of interferon-alpha (IFN-α) to restrain the growth and invasive potential of hepatocellular carcinoma (HCC) induced by hepatitis B virus (HBV) X protein. METHODS: The pcDNA3.1-HBx plasmid was transfected into Chang cells by Lipofectamine in vitro, and Chang/HBx was co-cultured with IFN-α. Cell survival growth curve and clonogenicity assay were used to test the growth potential of Chang/pcDNA3.1, Chang/HBx and IFN-α-Chang/HBx in vitro. Growth assay in nude mice was used to detect the growth potential of Chang/ pcDNA3.1, Chang/HBx and IFN-α-Chang/HBx in vivo. Wound healing and transwell migration assays were used to detect the invasive ability of Chang/pcDNA3.1, Chang/HBx and IFN-α-Chang/HBx. RESULTS: Compared with CCL13 cells transfected with pcDNA3.1, CCL13 with stable expression of hepatitis B virus X protein showed the characteristics of malignant cells with high capability of growth and invasion by detecting their growth curves, colony forming efficiency, wound healing , transwell migration assays and growth assays in nude mice. Its capability of growth and invasion could be controlled by IFN-α. CONCLUSION: IFN-α can restrain the growth and invasive potential of HCC cells induced by HBx protein, which has provided an experimental basis for IFN-α therapy of HCC.

Hepatitis B virus X protein accelerates the development of hepatoma

The chronic infection of hepatitis B virus(HBV) is closely related to the occurrence and development of hepatocellular carcinoma(HCC). Accumulated evidence has shown that HBV X protein(HBx protein) is a multifunctional regulator with a crucial role in hepatocarcinogenesis. However, information on the mechanism by which HBV induces HCC is lacking. This review focuses on the pathological functions of HBx in HBV-induced hepatocarcinogenesis. As a transactivator, HBx can modulate nuclear factor kappa-light-chain-enhancer of activated B cells(NF-κB) and transcription factor AP-2. Moreover, HBx can affect regulatory non-coding RNAs(ncRNAs) including microRNAs and long ncRNAs(lncRNAs), such as miRNA-205 and highly upregulated in liver cancer(HULC), respectively. HBx is also involved in epigenetic modification, including methylation and acetylation. HBx interacts with various signal-transduction pathways, such as protein kinase B/Akt, Wnt/β-catenin, signal transducer and activator of transcription, and NF-κB pathways. Moreover, HBx affects cellular fate by shifting the balance toward cell survival. HBx may lead to the loss of apoptotic functions or directly contributes to oncogenesis by achieving transforming functions, which induce hepatocarcinogenesis. Additionally, HBx can modulate apoptosis and immune response by direct or indirect interaction with host factors. We conclude that HBx hastens the development of hepatoma.

Hepatitis B virus X protein induces ALDH2 ubiquitin-dependent degradation to enhance alcoholic steatohepatitis

Background:Excessive alcohol intake with hepatitis B virus(HBV)infection accelerates chronic liver disease progression and patients with HBV infection are more susceptible to alcohol-induced liver disease.Hepatitis B virus X protein(HBx)plays a crucial role in disease pathogenesis,while its specific role in alcoholic liver disease(ALD)progression has not yet been elucidated.Here,we studied the role of HBx on the development of ALD.Methods:HBx-transgenic(HBx-Tg)mice and their wild-type littermates were exposed to chronic plus binge alcohol feeding.Primary hepatocytes,cell lines,and human samples were used to investigate the interaction between HBx and acetaldehyde dehydrogenase 2(ALDH2).Lipid profiles in mouse livers and cells were assessed by using liquid chromatography–mass spectrometry.Results:We identified that HBx significantly aggravated alcohol-induced steatohepatitis,oxidative stress,and lipid peroxidation in mice.In addition,HBx induced worse lipid profiles with high lysophospholipids generation in alcoholic steatohepatitis,as shown by using lipidomic analysis.Importantly,serum and liver acetaldehyde were markedly higher in alcoholfed HBx-Tg mice.Acetaldehyde induced lysophospholipids generation through oxidative stress in hepatocytes.Mechanistically,HBx directly bound to mitochondrial ALDH2 to induce its ubiquitin–proteasome degradation,resulting in acetaldehyde accumulation.More importantly,we also identified that patients with HBV infection reduced ALDH2 protein levels in the liver.Conclusions:Our study demonstrated that HBx-induced ubiquitin-dependent degradation of mitochondrial ALDH2 aggravates alcoholic steatohepatitis.

Dysregulation of β-catenin by hepatitis B virus X protein inHBV-infected human hepatocellular carcinomas

β-catenin is a key molecule involved in both cell-cell adhesion and Wnt signaling pathway.In our study,we found that,in the development of hepatocellular carcinoma(HCC),β-catenin was correlated with hepatitis B virus(HBV)X gene encoded protein,which is essential for HBV infectivity and is a potential cofactor in viral carcinogenesis.The expression levels of wild-typeβ-catenin and E-cadherin were decreased in HepG2 cells expressing hepatitis B virus X protein(HBx),accompanied by destabilization of adherens junction.Reverse transcrip-tase PCR(RT-PCR),Northern and Western blot showed that reduction of wild-typeβ-catenin expression involved degradation of the protein.However,RNA interference(RNAi)and luciferase assay indicated that HBx enhancedβ-catenin mediated signaling in HepG2 cells.In addition,immunohistochemical and Western blot analysis ofβ-catenin revealed that a decrease in theβ-catenin protein level was found in 58.3%of HBV-related HCCs versus 19.2%of non-HBV-related tumors.Our data suggest that the expression of HBx contributed to the development of HCC,in part,by repressing the wild-typeβ-catenin expression and enforcingβ-catenin-dependent signaling pathway,thus inducing cellular changes leading to acquisition of metastatic and/or proliferation properties.

Interaction of Hepatitis B Virus X Protein with the Pregnane X Receptor Enhances the Synergistic Effects of Aflatoxin B1 and Hepatitis B Virus on Promoting Hepatocarcinogenesis

Background and Aims:Hepatitis B virus(HBV)infection has been found to increase hepatocellular sensitivity to carcinogenic xenobiotics,by unknown mechanisms,in the generation of hepatocellular carcinoma.The pregnane X receptor(PXR)is a key regulator of the body’s defense against xenobiotics,including xenobiotic carcinogens and clinical drugs.In this study,we aimed to investigate the molecular mechanisms of HBV X protein(HBx)-PXR signaling in the synergistic effects of chemical carcinogens in HBV-associated hepatocarcinogenesis.Methods:The expression profile of PXR-cytochrome p4503A4(CYP3A4)signaling was determined by PCR,western blotting,and tissue microarray.Cell viability and aflatoxin B1(AFB1)cytotoxicity were measured using the cell counting kit-8 assay.Target gene expression was evaluated using transient transfection and real time-PCR.The genotoxicity of AFB1 was assessed in newborn mice with a single dose of AFB1.Results:HBx enhanced the hepatotoxicity of AFB1 by activating CYP3A4 and reducing glutathione Stransferase Mu 1(GSTM1)in cell lines.Activation of PXR by pregnenolone 16α-carbonitrile increased AFB1-induced liver tumor incidence by up-regulating oncogenic KRAS to enhance interleukin(IL)-11:IL-11 receptor subunit alpha-1(IL11RA-1)-mediated inflammation in an HBx transgenic model.Conclusions:Our finding regarding AFB1 toxicity enhancement by an HBx-PXR-CYP3A4/GSTM1-KRASIL11:IL11RA signaling axis provides a rational explanation for the synergistic effects of chemical carcinogens in HBV infection-associated hepatocarcinogenesis.

Expression of Cytokines in Mouse Hepatitis B Virus X Gene-transfected Model

The expression profile in the mouse hepatitis B virus X （HBx）-transfected model was investigated in order to lay a foundation for further study on the implication of cytokines expression in hepa- titis B virus （HBV） infection. Hydrodynamic injection method via the tail vein was used to establish the animal HBx-transfected model. By using microassay, the differential expression of gene in each group was analyzed, which was further confirmed by using real-time PCR and semi-quantitative PCR. Most of chemokine genes such as Cc12, Cc15, Cc19, MIG and IP-10 were up-regulated in the HBx-transfected mouse model versus the control mice, which was coincided with the microarray results. Western blotting and immunohistochemistry were applied to detect the expression of MIG and IP-10 in the liver tissues. Simultaneously, ELISA was adopted to measure the content of IFN-y in the liver tissues. DNA mi- croassay revealed that the expression of 611 genes changed in HBx-transfected mice as compared with that in pCMV-tag2B-transfected mice, and most of the screened chemokines were up-regulated （includ- ing MIG and IP-10）. Additionally, IFN-y protein levels were increased by 20.7% （P〈0.05） in pCMV-tag2B-HBx-transfected mice as compared with the untreated mice. IFN-7 protein levels were reduced by 53.9% （P〈0.05） in pCMV-tag2B-transfected mice as compared with the untreated mice, which was consistent with the up-regulation of MIG and IP-10. It was suggested HBx transfection could induce the expression of MIG and IP-10 in the liver tissues, which might play the roles in HBV-related liver immunity and cytokines-mediated antiviral effect.

Functional interaction of endoplasmic reticulum stress and hepatitis B virus in the pathogenesis of liver diseases

Hepatitis B virus(HBV) is a non-cytopathic virus that causes acute and chronic inflammatory liver diseases,often leading to the pathogenesis of hepatocellular carcinoma(HCC). Although many studies for the roles of HBV on pathogenesis of the liver diseases,such as non-alcoholic fatty liver disease(NAFLD),hepatic inflammation,cirrhosis,and HCC,have been reported,the mechanisms are not fully understood. Endoplasmic reticulum(ER) and mitochondria have the protective mechanisms to restore their damaged function by intrinsic or extrinsic stresses,but their chronic dysfunctions are associated with the pathogenesis of the various diseases. Furthermore,HBV can affect intraor extracellular homeostasis through induction of ER and mitochondrial dysfunctions,leading to liver injury. Therefore,the mechanism by which HBV induces ER or mitochondrial stresses may be a therapeutic target for treatment of liver diseases.

MicroRNAs as therapeutic strategy for hepatitis B virusassociated hepatocellular carcinoma: Current status and future prospects

Hepatocellular carcinoma(HCC)remains to be one of the top causing cancer-related deaths today.The majority of HCC cases are reported to be the result of chronic hepatitis B virus(HBV)infection.Current treatments for HBV-related HCC revolve around the use of drugs to inhibit viral replication,as a high level of viral load and antigen in circulation often presents a poor patient prognosis.However,existing therapies are inefficient in the complete eradication of HBV,often resulting in tumour recurrence.The involvement of microRNAs(miRNAs)in important processes in HBV-related HCC makes it an important player in the progression of HCC in chronic hepatitis B infected patients.In this review,we discuss the key aspects of HBV infection and the important viral products that may regulate cancerrelated processes via their interaction with miRNAs or their closely related protein machinery.Conversely,we also look at how miRNAs may go about regulating the virus,especially in vital processes like viral replication.Apart from miRNAs acting as either oncogenes or tumour-suppressors,we also look at how miRNAs may function as biomarkers that may possibly serve as better candidates than those currently employed in the diagnosis of HBV infection or HBV-related HCC.A summary of the roles of miRNAs in HBV-related HCC will hopefully lead to a gain in understanding of the pathogenesis process and pave the way for new insights in medical therapy.

Hepatitis B virus infection and the risk of hepatocellular carcinoma

Epidemiological studies have provided overwhelming evidence for a causal role of chronic hepatitis B virus(HBV) infection in the development of hepatocellular carcinoma(HCC).However,the pathogenesis of HBV infection and carcinogenesis of HBV-associated HCC are still elusive.This review will summarize the current knowledge on the mechanisms involved in HBV-related liver carcinogenesis.The role of HBV in tumor formation appears to be complex,and may involve both direct and indirect mechanisms.Integration of HBV DNA into the host genome occurs at early steps of clonal tumor expansion,and it has been shown to enhance the host chromosomal instability,leading to large inverted duplications,deletions and chromosomal translocations.It has been shown that the rate of chromosomal alterations is increased significantly in HBV-related tumors.Prolonged expression of the viral regulatory HBV x protein may contribute to regulating cellular transcription,protein degradation,proliferation,and apoptotic signaling pathways,and it plays a critical role in the development of hepatocellular carcinoma.

Long noncoding RNAs in hepatitis B virus replication and oncogenesis

Several diverse long noncoding RNAs(lncRNAs)have been identified to be involved in hepatitis B virus(HBV)replication and oncogenesis,especially those dysregulated in HBV-related hepatocellular carcinoma(HCC).Most of these dysregulated lncRNAs are modulated by the HBV X protein.The regulatory mechanisms of some lncRNAs in HBV replication and oncogenesis have been characterized.Genetic polymorphisms of several lncRNAs affecting HBV replication or oncogenesis have also been studied.The prognosis of HCC remains poor.It is important to identify novel tumor markers for early diagnosis and find more therapeutic targets for effective treatments of HCC.Some dysregulated lncRNAs in HBV-related HCC may become biomarkers for early diagnosis and/or the therapeutic targets of HCC.This mini-review summarizes these findings briefly,focusing on recent developments.

HBx activates FasL and mediates HepG2 cell apoptosis through MLK3-MKK7-JNKs signal module

AIM： To investigate the possible mechanism by which hepatitis B virus X protein （HBx） mediates apoptosis of HepG2 cells. METHODS： HBx expression vector pcDNA3.1-X was transfected into HepG2 cells to establish an HBx high- expression cellular model as pcDNA3.1-X transfected group. The pcDNA3.1-X and pSilencer3.1-shHBX （HBx antagonist） were cotransfected into HepG2 cells to es- tablish an HBx low-expression model as RNAi group. Untransfected HepG2 cells and HepG2 cells transfected with negative control plasmid were used as controls. Apoptosis rate, the expression of Fas/FasL signaling pathway-related proteins and the phosphorylation lev- els of MLK3, MKK7 and JNKs, which are upstream molecules of death receptor pathways and belong to the family of mitogen-activated protein kinases （MAPKs）,were measured in each group RESULTS： Compared with HepG2 cell group and RNAi group, apoptosis rate, the expression of Fas and FasL proteins, and the activation of MLK3, MKK7 and 3NKs were increased in the pcDNA3.1-X transfected group. The activation of JNKs and expression of FasL protein were inhibited in the pcDNA3.1-X transfected group when treated with a known JNK inhibitor, SP600125. When authors treated pcDNA3.1-X transfected group with K252a, a known MLK3 inhibitor, the activation of MLK3, MKK7 and 3NKs as well as expression of FasL protein was inhibited. Furthermore, cell apoptosis rate was also significantly declined in the presence of K252a in the pcDNA3.1-X transfected group. CONCLUSION： HBx can induce HepG2 cell apoptosis via a novel active MLK3-MKK7-JNKs signaling module to upregulate FasL protein expression.

Integrated analysis of micro RNA and m RNA expression profiles in HBx-expressing hepatic cells

AIM To identify the miRNA-mRNA regulatory network in hepatitis B virus X (HBx)-expressing hepatic cells. METHODS A stable HBx-expressing human liver cell line L02 was established. The mRNA and miRNA expression profiles of L02/HBx and L02/pcDNA liver cells were identified by RNA-sequencing analysis. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis was performed to investigate the function of candidate biomarkers, and the relationship between miRNA and mRNA was studied by network analysis. RESULTS Compared with L02/pcDNA cells, 742 unregulated genes and 501 downregulated genes were determined as differentially expressed in L02/HBx cells. Gene ontology analysis suggested that the differentially expressed genes were relevant to different processes. Concurrently, 22 differential miRNAs were also determined in L02/HBx cells. Furthermore, integrated analysis of miRNA and mRNA expression profiles identified a core miRNA-mRNA regulatory network that is correlated with the carcinogenic role of HBx. CONCLUSION Collectively, the miRNA-mRNA network-based analysis could be useful to elucidate the potential role of HBx in liver cell malignant transformation and shed light on the underlying molecular mechanism and novel therapy targets for hepatocellular carcinoma.

Genotype-dependent activation or repression of HBV enhancer Ⅱ by transcription factor COUP-TF1

AIM: To study the expression of HBV enhancer Ⅱ by transcription factor COUP-TF1. METHODS: In order to study the regulation of HBV variants in the vicinity of the NRRE we cloned luciferase constructs containing the HBV enhancer Ⅱ from variants and from HBV genotypes A and D and cotransfected them together with expression vectors for COUP-TF1 into HepG2 cells. RESULTS: Our fi ndings show that enhancer Ⅱ of HBV genotype A is also repressed by COUP-TF1. In contrast, two different enhancer Ⅱ constructs of HBV genotype D were activated by COUP-TF1. The activation was independent of the NRRE because a natural variant with a deletion of nt 1763-1770 was still activated by COUP- TF1. CONCLUSION: Regulation of transcription of the HBV genome seems to differ among HBV genomes derived from different genotypes. These differences in transcriptional control among HBV genotypes may be the molecular basis for differences in the clinical course among HBV genotypes.

Hepatitis B Virus Induces Microtubule Stabilization to Promote Productive Infection through Upregulating Microtubule-associated Protein 1S

Background and Aims:Continuous release and transmission of hepatitis B virus(HBV)is one of the main factors leading to chronic hepatitis B(CHB)infection.However,the mechanism of HBV-host interaction for optimal viral transport is unclear.Hence,we aimed to explore how HBV manipulates microtubule-associated protein 1S(MAP1S)and microtubule(MT)to facilitate its transport and release.Methods:The expression of MAP1S or acetylated MT was investigated by immunofluorescence,RT-PCR,immunoblotting,and plasmid transfection.MAP1S overexpression or knockdown was performed by lentiviral infection or shRNA transfection,respectively.HBV DNA was quantified using q-PCR.Results:Significantly higher level of MAP1S in HepG2215 cells compared with HepG2 cells was detected using RT-PCR(p<0.01)and immunoblotting(p<0.001).Notably,stronger MAP1S expression was observed in the liver tissues of patients with CHB than in healthy controls.MAP1S overexpression or knockdown demonstrated that MAP1S promoted MT acetylation and reduced the ratio of HBV DNA copies inside to outside cells.Further,transfection with the hepatitis B virus X protein(HBx)-expressing plasmids induced significantly higher level of MAP1S than that in controls(p<0.0001),whereas HBVX−mutant-encoding HBV proteins(surface antigen,core protein,and viral DNA polymerase)hardly affected its expression.Conclusions:These results demonstrate that HBx induces the forma tion of stable MTs to promote the release of HBV particles through upregulating MAP1S.Thus,our studies delineate a unique molecular pathway through which HBV manipulates the cytoskeleton to facilitate its own transportation,and indicate the possibility of targeting MAP1S pathway for treatment of patients with CHB.