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 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.

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.

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.