Hepatitis B Virus Mediated Regulation On FOXP3and TIM-3in HCC

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the third most common cause of cancer mortality.80%of HCCs are linked to chronic infections with the hepatitis B virus (HBV) or hepatitis C virus. China is one of the HCC high-incidence districts which takes up55%of HCC cases in the world. In recent years, HCC incidence in developed countries is increasing abruptly. Chronic hepatitis B virus infection has been identified as a leading risk factor for HCC in China. So it is definitely urgent to explore the accurate mechanisms of HBV associated liver diseases.The mechanism of HBV related HCC is complicated. The integration of HBV-DNA into the host genome can trigger cis-activation directly. The regulatory proteins HBx and preS2encoded by integrated subviral HBV genomes can exert a tumor promoter-like function, resulting in positive selection of cells producing a functional regulatory protein. To search the key genes regulated by HBV, not only provides newsights into mechanisms of HBV related HCC development, but also gives new potential targets for drug design.Forkhead box protein3(FOXP3) and T cell immunoglobulin and mucin-domain-containing molecule3(TIM-3) are both newly reported important immune regulatory molecules. Our previous work indicated that FOXP3/TIM-3expression was increased on peripheral blood mononuclear cells (PBMCs) and hepatic mononuclear cells (HMCs) from chronic hepatitis B (CHB) patients. HBV transfection up-regulated TIM-3expression on NK92cells which reversly inhibites cytotoxicity of NK cells. All above strongly suggested that HBV mediated FOXP3/TIM-3would contribute to the development of HBV associated liver diseases. In this study, we aim to investigate the expression and functional role of FOXP3/TIM-3in hepaocellular carcinoma cells and further explore the mechanism of HBV meidated upreguation of FOXP3/TIM-3. We focus our reseach on the following aspects: PART I HBV mediated FOXP3upregulation in malignant hepatocye and its roles in the development of HCCPART II HBV-mediated TIM-3upregulationPART I HBV mediated FOXP3upregulation in malignant hepatocytes and its role in the development of HCCTranscription factor forkhead box protein3(FOXP3) has been widely known as the master regulator for the development and function of regulatory T cells (Tregs) and the only definitive marker of CD4+CD25+Tregs. The loss of FOXP3leads to the lack of Tregs, resulting in fatal autoimmunity, whereas overexpression of FOXP3results in severe immunodeficiency. Genome-wide analysis reveals that FOXP3is able to bind to about700genes, suggesting that it acts as both a transcriptional activator and a repressor. FOXP3can form a complex with transcription factors and regulate the transcription of several target genes. This confers to the immune suppressive phenotypes of Tregs. Increased frequencies of FOXP3+Tregs have been reported in different types of cancers, including hepatocellular carcinoma (HCC). Multivariate analysis reveals that the prevalence of Tregs infiltration is an independent prognostic factor in HCC.As reported, FOXP3expression was initially thought to be restricted in the lymphocytic linage. However, recent reports described the FOXP3expression on both normal and cancerous cells in addition to Tregs. FOXP3expression can be detected on epithelial breast and prostate cells. FOXP3expression was extensive down-regulated among breast and prostate cancer tissues in which FOXP3works as a cancer suppressor by down-regulating the expression of oncogenes. In contrast, several other reports described the augmented expression of FOXP3in tumour cells, such as melanoma and pancreatic carcinoma cells. Interestingly, FOXP3expression in pancreatic cancer cells enables them to inhibit the proliferation of T cells, suggesting that FOXP3expression in cancer cells might trigger a mechanism of tumour immune evasion. Collectively, FOXP3can be expressed in solid tumour cells and its expression profile and function might completely contradict in different types of tumours due to distinct biological characteristics. This contradiction strongly suggests a careful caution when FOXP3is used as an indicator of cancer risk in humans. Thus, it is urgent to explore the expression pattern and function of FOXP3in different types of tumours.This study was designed to explore the expression and biological functions of FOXP3in HCC and to uncover the role of HBx in regulating FOXP3expression. Our data here, for the first time, identified new mechanisms of FOXP3in the development of HBV related HCC.Objectives1To detecte the expression profiles of FOXP3in different cells in HCC;2To uncover the role of HBV and its coding proteins in regulating FOXP3expressionand the molecular mechanism;3To explore the biological functions of FOXP3expressed in HCC cell lines.Methods1Detection of the expression profiles of FOXP3in different cells in HCC1.1Tissues and immunohistochemical staining10tissue samples, collected from patients with HCC from Shandong Provincial Hospital, and tissue array including43liver cancer tissues and41matched adjacent normal tissues were invloved in immunohistohemical staining with anti-FOXP3antibody.1.2Cell lines and HCC tissues for western blot and RT-PCRHuman HCC cell lines Be17402, QSG7701, HepG2, Huh-7, SMMC7721, HepG2.2.15and immortal liver cell line L02cells were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences. HepG2.2.15cells harbor4copies of HBV DNA and expressing all viral proteins.4tissue samples were collected from patients with HCC from Shandong Provincial Hospital.1.2.1Reverse transcriptase polymerase chain reaction (RT-PCR)Total RNA was extracted using Trizol reagent from cells and tissues. Reverse RT-PCR were performed as previously report.1.2.2Western blot analysisLysates were obtained from HCC tissues or106cells. Immunoblotting was performed with anti-FOXP3, anti-cyclin E, anti-cyclin A, anti-CDK4, anti-P27, anti-c-Jun and anti-β-actin.1.3Immunofluorescence analysis with confocol microscopeImmunofluorescence analysis was used to analyze the FOXP3protein distribution in HCC cell line HepG2, breast cancer cell line MCF-7and prostate cancer cell line PC3. Cells grown on coverslips were fixed in4%paraformaldehyde, and then were immunostained with anti-FOXP3antibody, followed by detection with rhodamine-conjugated secondary antibody. Nuclear staining with DAPI was performed after incubation with secondary antibodies.2Study of the role of HBV and its coding proteins in regulating FOXP3expression and the molecular mechanisms2.1Plasmids A series of truncated human FOXP3promoter-reporter constructs were generously provided by Dr. Schmidt-Weber, C. B. Four truncated FOXP3promoter-reporter plasmids were constructed by inserting PCR products of FOXP3into KpnI/XhoI sites of pGL4.10, respectively named pGL-445/176, pGL-423/176, pGL-401/176and pGL-377/176. PGL-464mut was constructed with site-directed mutation of the AP-1binding site by a PCR-based protocol.2.2Transfection and luciferase assayTransfections or cotransfections were performed with FuGENE(?) HD transfection reagent for HepG2or lipofectamineTM2000for other cell lines. Luciferase reporter assay was performed to analyze the promoter activities.2.3Electrophoretic mobility shift assay (EMSA)Nuclear extracts were prepared from HepG2cells transfected with HBx. Dephosphorylated, gel-purified20bp fragment (-465/-445bp, probe), fragment (-465/-445bp) with mutation at predicted AP-1binding site (mut probe) and a classical AP-1binding site were end-labeled with digoxigenin (indicated as*) and used as probe. EMSA was performed according to mannual. Non-labeled self oligonucleotide (probe), nonspecific SP-1binding sequence (non-SP-1), classical AP-1binding site (Wt AP-1) or mutant AP-1binding site was involved separately as the competitor. The antibody against HA tag was used to super shift the binding.3The biological functions of FOXP3involved in HCC3.1Cell proliferation and colony formation analysisHepG2or HepG2.2.15cells transfected with pCI-hFOXP3or si-FOXP3. The Cell Counting Kit-8(CCK-8) was used to measure cell proliferation with all enzyme-labeled minireade at A450respectively on1-7day. For the soft agar colony formation assay, after transfected, cells were seeded in six-well plate and cultured for7days. Colonies were stained with gentian violet and counted under a microscope. Three to five wells were analyzed for each experiment.3.2Cell cycle analysisHepG2or HepG2.2.15cells transfected with pCI-hFOXP3or si-FOXP3for48hours were harvested and stained with15μM of PI. The cell cycles were detected using flow cytometry. The percentages of the nuclei at each phase of the cell cycle were analyzed using the MultiCycle software program.3.3Isolation of T cellsPeripheral blood mononuclear cells (PBMC) were isolated from human peripheral blood from healthy donors by Ficoll-Hypaque gradient centrifugation. CD4+T cells were isolated, followed by magnetic separation on MACS columns.3.4Coculture experiments and carboxyfluorescein succinimidyl esterproliferation (CFSE) assaysHepG2cells were cocultured with2×106CD4+T cells for3days. T cells were labeled with CFSE. Proliferation of T cells was measured by flow cytometry on the basis of CFSE dilution and calculated as the ratio of the number of proliferating over the total number of T cells.4Statistical AnalysisComparisons of quantitative data were analyzed by Student t test between two groups. The statistical correlation between the clinical parameters of HCC and the FOXP3staining level of tissue array was analysed by the Chi-square test or Fisher's exact test as appropriate. Ap<0.05was accepted as a significant criterion.RESULTS1FOXP3expression in HCC tissues and cell lines1.1FOXP3expression can be detected on hepatocytes besides lymophcytes in HCC tissuesWe detected FOXP3staining in HCC tissues and HCC tissue array. FOXP3staining can be detected in hepatic cells, Kuppfers cells, intrahepatic cholangio cells and tumor infiltrated lymphoctes (TILs). FOXP3protein can be detected in all4tumour tissues while only in2out of4paratumour tissues.1.2Augment FOXP3expression in malignant hepatocytes with HBVRT-PCR and western blot confirmed the FOXP3expression in all detected HCC cell lines while the FOXP3expression in immortal liver cell line L02cells was comparatively low. It is interesting to see that the FOXP3expression in HepG2.2.15cells which harbors HBV in its genome was comparatively higher than that in the HepG2cells. Consistently, immunohistochemical staining showed a significant correlation between HBV infection and FOXP3expression in180cores tissue array.1.3Subcellular distribution of FOXP3in different cellsImmunohistochemical staining and confocol assay demonstrated that FOXP3could be detected both in nuclei and cytoplasm, however, the subcellular distribution of FOXP3in hepatocytes from cancer and paracancer sections were different. All most all FOXP3was stained in cytoplasm in cancer sections while FOXP3located in the nuclear in paracancer sections.2FOXP3expressin can be upregulated by HBV and its encoding proteins2.1HBV and its encoding proteins up-regulates FOXP3expression in HCC cell linesRT-PCR and western blot results demonstrated that transfection of HBV or its encoding proteins led to significant increased FOXP3expression in both HepG2and BeI7402cells. HBx and preS2overexpression showed the greatest upregulation effects on FOXP3expression. In consistence, knockdown of HBV genes,especially HBx and preS2by asons in HepG2.2.15cells greatly reduced FOXP3expression. These indicated that FOXP3can be upregualted by HBV and its encoding proteins.2.2Augmented FOXP3expression in HBV-DNA positive PBMC from CHB patientsIn order to study the correlation of FOXP3expression with HBV in vivo, PBMC were collected from patients with CHB. HBV-DNA was detected in PBMC by PCR analysis which was used to divide CHB samples into two groups:HBV positive and HBV negative. Real time RT-PCR analysis showed increased FOXP3expression in PBMC with HBV-DNA than that without HBV DNA, which is consistent with in vitro overexpression and knockdown assays,3HBV transactivates FOXP3promoter in hepatoma cells3.1The FOXP3promoter region contains cell-specific activityFOXP3promoter displayed significantly higher activities in HBV positive HepG2.2.15cells than in those of HBV-negative HepG2and Bel7402cells.The core promoter activity located at-465/176.3.2HBV especially HBx and preS2significantly up-regulate FOXP3promoter activity:Overexpression of HBV significantly up-regulated FOXP3promoter activity of pGL-465/176in HepG2and Bel7402cells, especially HBx and preS2. The up-regulation of FOXP3promoter activity occurred in an dose-dependent manner. On the contrary, asons for HBx/preS2/HBc treatment decreased the promoter activity of pGL-465/176in HepG2.2.15cells in a dose dependant manner. These data indicate that HBx and preS2transcriptionally upregulates FOXP3expression in HCC cells.3.3HBx transcriptionally activates FOXP3through AP-13.3.1The AP-1binding site plays a critical role in HBx-mediated FOXP3transcriptional activation EMSA assay results indicate that the AP-1binding site within the20bp region from-465bp to-445bp plays a critical role in HBx-mediated FOXP3transcriptional activation. EMSA using nuclear extracts from HepG2cells transfected with pc-HBx-HA further verified this hypothesis.3.3.2HBx transactivated FOXP3in an AP-1dependant manner Knockdown of AP-1by si-c-Jun destroyed the HBx-mediated FOXP3up-regulation, which further validated the hypothesis that HBx transactivating FOXP3is AP-1dependant.4FOXP3promotes cell growth of hepatocellular carcinoma cell lines4.1FOXP3promoted cell growth and colony forming ability Overexpressed FOXP3significantly promoted cell growth and colony forming ability, while FOXP3knockdown reduced cell growth and colony formation.4.2FOXP3promoted HCC cells entering S-phase cell cycle and influedced the cyclins Cell cycle analysis showed a higher cell proportion in S phase in HepG2cells transfected with FOXP3while si-FOXP3decreased cell proportion of S phase in HepG2.2.15cell. Consistently, FOXP3-transfection significantly up-regulated the level of cyclin E, cyclin A and CDK4protein but not the P27protein in HepG2and HepG2.2.15cells.4.3FOXP3expression in HepG2cells inhibits CD4+T-cell proliferation in vitroFOXP3overexpression in HepG2cells strongly strengthened the inhibition on the proliferation of CD4+T cells. This suggests that expression of FOXP3endow hepatoma cells to suppress T cells in HCC.PART II HBV mediated TIM-3regulation in HCCT-cell immunoglobulin-and mucin-domain-containing molecule-3(TIM-3) was the first, and hitherto the only, surface molecule that can specifically identify Thl cells from Th2cells in both mice and human. Engagement of TIM-3by its ligand galectin-9negatively regulates IFN-y secretion and blocks the induction of T cell tolerance. Accumulated evidences support that TIM-3is expressed not only on Thl cells, but also on many other immune cells and induces distinct signalling events which may influence a range of inflammatory conditions, including chronic virus infection, diabetes and multiple sclerosis. Wiener Z. recently identified TIM-3expression on both mast cells and tumour cells in melanoma tissue sections. TIM-3can be up-regulated by TGF-β treatment and contribute to the lower adhering capacity of tumour cells which may support the survival of melanoma.In this study, we detected TIM-3expression on PBMC,tumor cells and tumor infiltration lymphocytes (TILs) in CHB and HCC patients. The expression levels of TIM-3show significant correlation with HBV infection. The role of HBV in transregulating TIM-3was studied。Objectives1Detected the expression profiles of TIM-3in different cells in HCC; 2Uncover the role of HBV and its coding proteins in regulating TIM-3expression and the molecular mechanism.Methods1Expression profiles of TIM-3in different cells in HCC1.1Immunohistochemical staining Liver tissues from50HCC patientswere obtained to detect TIM-3expression by immunohistochemical staining.1.2Isolation of PBMCs Same as PART I.1.3Overexpression and block of HBV in cell lines Plasmid constructs of HBV and its coding proteins and asons designed to block HBV coding proteins were transfected to cell lines using FuGENE(?) HD transfection reagent for HepG2or lipofectamineTM2000for other cell lines.2Uncover the role of HBV and its coding proteins in regulating TIM-3expression and the molecular mechanism2.1Constructs of TIM-3promoter A series of truncated TIM-3promoter reporter plasmids were constructed by inserting PCR products of FOXP3into pGL4.10, respectively named pGL-2083/242,-1546/242,-1171/242,-639/242,-318/242,-253/242,-231/242,-88/242.2.2Transfection and luciferase assay:Same as PART IRESULTS1Expression profile of TIM-3in different cells in HCC and augmented by HBV1.1The PCR assay reveals the detection of HBV-DNA expression which was used to divide CHB samples to two groups (positive or negative). Augmented FOXP3expression was observed in HBV-DNA positive group.1.2Immunohistochemical staining demonstrated that TIM-3was stained in cytoplasm in cancer sections.1.3RT-PCR and western blot results demonstrated that transfection of HBV or HBx led to significant increased TIM-3expression in both HepG2and Be17402cells; in consistence, knockdown of HBV by asons in HepG2.2.15cells greatly reduced FOXP3expression.2HBV transactivates the activity of TIM-3promoter in different cell lines in vitro2.1TIM-3promoter displayed activities in both B16and HCC cell lines, while activities were higher in HBV positive HepG2.2.15cells than in those of HBV-negative Be17402cell.2.2Overexpression of either HBV or its coding proteins up-regulated TIM-3promoter activity of pGL-231/242in HepG2and Bel7402cells. The up-regulation of TIM-3promoter activity occurred in an HBx dose-dependent manner. On the contrary, as-preS2treatment decreased the promoter activity of pGL-231/242in HepG2.2.15cells in a dose dependant manner. These data indicate that HBx transcriptionally upregulates TIM-3expression in HCC cells.Conclusions1. Immunoregulatory molecules FOXP3/TIM-3were expressed in hepatic cells by cell lines and clinical tissues detection, while expression level corelated with HBV infection;2. Augmented expression of FOXP3/TIM-3in hepatoma cells mediated by HBV was demonstrated by overexpression and knockdown assay in vitro;3. HBV transactivates the activity of FOXP3/TIM-3promoter in hepatoma cells by dual-luciferase assay; while HBx transactivating FOXP3was in an AP-1dependant manner;4. FOXP3promotes cell growth of hepatocellular carcinoma cell lines, contributing to HCC development and immune escape.Innovations and significance1. In this study, we firstly reported the increased expression of FOXP3/TIM-3on HCC cells mediated by HBV infection;2. FOXP3expression in tumours displayed controversial biofunctions. We demonstrated that FOXP3promoted cell growth of hepatocellular carcinoma cell lines, contributing to HCC development and immune escape;3. We explored the accurate mechanisms of HBx transactivating FOXP3in an AP-1site dependent manner;4. HBV infection was involved in HCC partialy through regulating TIM-3expression.