| ObjectiveHepatocellular carcinoma is one of the major diseases that endanger human health.Its mortality rate is only behind lung cancer in the world.HCC is a typical inflammatory related tumor,usually developed by patients with chronic hepatitis.The inflammatory microenvironment of chronic inflammation is closely related to the development of hepatocellular carcinoma.Professor Lieping Chen proposed the "adaptive resistance hypothesis" of tumor immune escape.The hypothesis points out that tumor is selected by the anti-tumor immune response to have resistant ability to the immune response.The development process of tumor is a few years or even decades.T lymphocytes in tumor tissue secrete cytokines such as IFN-?.IFN-? can enhance the activity of immune cells,thereby killing tumor cells.But IFN-? can also enhance expression of B7-H1 molecules in tumor cells at the same time.B7-H1 molecules in the tumor cell surface combined with PD-1 expressed in the T cell surface.PD-1 is an important immunosuppressive transmembrane protein and expressed on the surface of T cells.B7-H1 binds to PD-1 to inhibit T cell activation and cytokine production,leading to exhaustion depletion of T cell function,which in turn promotes tumor cells immune escape.With the continuous development of immunotherapy progress,especially in recent years,the rapid development of immunological checkpoint therapy,human treatment of the tumor has made great progress.The latest generation of immunological checkpoint therapy plays a therapeutic role by blocking the interaction of PD1 on lymphocytes with B7-H1 molecules on antigen-presenting cells or tumor cells.FDA has approved drugs for PD1-PDL1 signaling pathway for clinical treatment of non-small cell lung cancer and bladder cancer.The remaining indications are in the clinical trial phase.Tumor Microenvironment(TME)is a complex environment for tumor cells to survive,mainly composed of a variety of cells and a large number of cytokines.The expression of B7-H1 is induced by a variety of cytokines,such as IFN-??TNF-??IL-4?VEGF and so on.IFN-? and TNF-? are two major cytokines.So we ask how IFN-? induced B7-H1 expression in HCC cells.Does TNF-? can cause elevated B7-H1 expression on HCC cells?And what is the relationship between the two cytokines in the induction of B7-HI1 expression?Does the inducible expression of B7-H1 molecule has a function of immune resistance?In order to solve the above problems we conducted the following research.In this study,we first studied that IFN-? can induce the expression of B7-H1 on the surface of HCC cells,mainly through JAK/STAT1 signaling pathway.TNF-? and IFN-? can synergistically promote the expression of B7-H1 molecules.To further study the mechanism of synergistic effect,it was found that TNF-? promoted the expression of IFNGR1 and IFNGR2 through NF-?B signaling pathway,and then promoted B7-H1 expression induced by IFN-? in the HCC cells.In order to further study whether the high expression of B7-H1 on the surface of HCC cells induced by TNF-? and IFN-?has a function,we have carried out co-culture experiments and animal experiment.Experiment shows that TNF-? and IFN-? induced B7-H1 expression on the surface of HCC cells inhibit T cells proliferation.Animal experiment shows that synergistically induced B7-H1 molecules can promote the immune escape of tumor cells.Methods1.IFN-y induced the expression of B7-H1 on HCC cells IFN-y-induced expression of B7-H1 on the surface of HCC cells in a concentration and time dependent mannerSMMC-7721 was seeded on the plate.The expression of B7-H1 was induced by different concentrations of IFN-?.IFN-? with a storage concentration of 100ng/?l was diluted.And the concentration was 100ng/ml,50ng/ml,20ng/ml and lOng/ml.The expression of B7-H1 RNA was detected by RT-PCR after 24h.The expression of B7-H1 was induced by IFN-y(50 ng/ml)at the same concentration for 0,1,2,3,6,12 and 24h respectively.The expression of B7-H1 RNA was detected by RT-PCR and qPCR.IFN-? induced expression of B7-H1 molecule on the surface of HCC at RNA and protein levelsHCC cell lines SMMC-7721,HLE-7402,HuH-7 and Hep3B were purchased from the Chinese Academy of Sciences Shanghai cell bank.The expression of B7-H1 RNA was detected by RT-PCR in the presence of 50ng/ml IFN-y for 6 h.We selected two of the cell lines SMMC-7721 and HuH-7 follow-up experiments.The expression of B7-H1 RNA was further detected by the qPCR method.The expression of B7-H1 protein was detected by flow cytometry.After induction with 50ng/ml IFN-y for 24 h.Plate was washed once with PBS to remove dead cells and then cells will be digested into a single cell by trypsin.Cells were labeled human B7-H1 flow antibody.2.IFN-?-induced expression of B7-H1 on the surface of HCC cells was mainly mediated by JAK/STAT1 signaling pathway.(1)First,to detect the signal pathway involved in the expression of B7-H1 in HCC induced by IFN-y(50ng/ml).The activation of STAT1,AKT,P65,P38,MEK,JNK signaling pathway was detected by Western-blot at 0,5,15,30 and 60 min.(2)IFN-y induced expression of B7-H1 signaling in HCC cells were NF-?B,PI3K,MEK,JNK,P38,JAK/STAT1.Buy a variety of signal pathway inhibitors,Western-blot was used to detect the inhibitory effect of various inhibitors on the corresponding signal pathway and the optimal inhibitory concentration(BAY11-7082(NF-?B),LY294002(PI3K?/?/?),U0126(MEK1/2),SP600125(JNKl/2/3),SB202190(p38?/?),Ruxolitinib(JAK1/2)).SMMC-7721 was seeded on the plate.Western-blot was used to detect the inhibitory effect.SMMC-7721 was seeded on the plate,add the corresponding inhibitor 1h ahead.Then add IFN-y(50ng/ml)at the same time containing a new inhibitor,culture 15min.The expression of B7-H1 was detected by qPCR method after 6 hours.(3)The above results show that IFN-y induced expression of B7-H1 on the surface of HCC was mainly mediated by JAK/STAT1 signaling pathway.We synthesized two important transcription factors STAT1 and IRF1.First verify the effect of interference small interference.SMMC-7721 was seeded on the plate.The next day was grown to about 70%for transfection.Change new liquid after 6h,transfection 24h collection RNA.RT-PCR was used to detect the expression of STAT1 and IRF1.SMMC-7721 cells were transfected with STAT1 and IRF1 after 24h.Cells were stimulated with 50 ng/ml of IFN-y for 6h.QPCR was used to detect B7-HI RNA expression.3.The synergy of TNF-? and IFN-y in the induction of B7-H1 expression in the HCC cellsWe asked whether TNF-a could have effect on the induction of B7-H1 expression by IFN-y in HCC cells.SMMC-7721 cells,HuH-7 cells were seeded on the plate.The expression of B7-H1 was induced by TNF-a(50ng/ml),IFN-y(5ng/ml)or both.The expression of B7-H1 in RNA level was detected by qPCR after 6 hours induction.Flow cytometry method to detect changes in protein levels after 24 hours induction.4.The mechanism of the synergy of TNF-a and IFN-y in induction of B7-H1 expression in the HCC cells(1)To analyze the effect of TNF-? on IFN-y signaling pathwayThe changes of signal pathway in B7-H1 expression induced by TNF-a and IFN-y were detected by using various inhibitors of signal pathway.SMMC-7721 cells and HuH-7 cells were treated with inhibitors.The cells were induced to express B7-H1 by TNF-a(50ng/ml)and IFN-y(5ng/ml)containing the new inhibitor medium,RNA collection after 6h.The expression of B7-H1 was detected by qPCR method.In order to further verify the signal pathway changes,we use small interfering RNA for signaling pathways siSTAT1,siIRF1,siP65,sic-JUN,sic-FOS and siATF2.Change new liquid after 6h.After transfection of small interfering RNA 24h,induced B7-H1 expression by TNF-?(50ng/ml)and IFN-?(5ng/ml),after 6h RNA collection,the changes of B7-H1 were detected by qPCR method.The cells were incubated with TNF-?(50ng/ml)and IFN-?(5ng/ml)medium,after 24h collection cell protein.Western-blot was used to detect the changes of JAK2 and p-JAK2,STAT1 and p-STAT1 in JAK/STAT1 signaling pathway.(2)Effects of TNF-? on the expression of IFNGR1 and IFNGR2While testing the JAK/STAT1 signaling pathway upstream molecules IFNGR1?IFNGR2 changes in the situation.Cells were incubated with TNF-?(50 ng/ml)and IFN-?(5 ng/ml)to induce expressing B7-H1.RNA collection after 6h.QPCR method to detect changes in IFNGR1?IFNGR2.In order to further study the mechanism of TNF-? enhancing the expression of IFNGR1 and IFNGR2.According to the literature,TNF-? major signaling pathways are NF-?B,MEK,P38,JNK and so on.First use the signal pathway inhibitors.SMMC-7721 cells and HuH-7 cells were seeded on the plate.1h ahead to add the inhibitors,(5?M BAY11-7082(NF-?B),5?M U0126(MEKl/2),5?M SB202190(p38a/?),5?M SP600125(JNK1/2/3)),qPCR method to detect changes in IFNGR1,IFNGR2.Further detect the role of NF-?B signaling pathway.SMMC-7721 cells and HuH-7 cells were seeded on the plate and transfected with P65 small interfering RNA.Induced with TNF-? for 6h,and qPCR method to detect changes in IFNGR1,IFNGR2.5.B7-H1 expression induced by the combination of TNF-? and IFN-? had immune resistance functionIn order to test whether TNF-? and IFN-? induce the expression of B7-H1 in HCC cells has a function.We conducted a co-culture experiment and animal experiment.(1)Co-culture experiment of murine tumor cells and T cells a).The synergy of IFN-? and TNF-? in the induction of B7-H1 expression in the murine liver cancer cellsMice liver cells Hepal-6 was seeded on the plate.The expression of B7-H1 was induced by IFN-?,TNF-? or IFN-? and TNF-?.QPCR was used to detect the expression of B7-H1 on Hepal-6 cells.IFN-?,TNF-? concentration and the same method as described above,after 24h,Wash the cells once with PBS to remove dead cells,and then labeled with mouse B7-H1 flow antibody.b).siRNA assayAccording to the experimental we synthesized mice B7-H1 small interference.we detection of small interference RNA effect.Hepal-6 cell was seeded on the plate,1.8×105 cells per well,the second day of cell growth to 70%of the transfected mice B7-H1 small interference.c).Treatment with IFN-? and TNF-?IFN-? storage concentration of 50ng/?l,IFN-y concentration of action 5ng/ml,Take 2?l of the stock solution in 18?l of T cell culture medium and dilute the concentration of 5ng/?l.Add 1?l of IFN-? dilution to I ml of medium.TNF-? storage concentration of 100ng/?l,TNF-? concentration of action 50ng/ml,Take 8?l of the stock solution in 72?l of T cell culture medium,dilute the concentration of 10ng/ul.Add 5?l of TNF-? dilution to 1 ml of medium.Hepal-6 cells were transfected for 6 h,and then addition of IFN-? and TNF-?.d).Treated with mitomycinCOn the third day,the tumor cells were treated with mitomycinC.MitomycinC added 747.65?l DMSO is the concentration of 20mM(6.687?g/?l).The optimal concentration of mitomycinC was added to the tumor cell culture,37? for 30min,and wash three times removed the residual mitomycin C.e).PBMCOn the third day,the C57BL/6J SPF grade 6-8 week old mice were sacrificed.The dead mouse in 75%alcohol for 5 min.The spleen tissue of the mice was taken out in the clean bench.The cell suspension was transferred to a 10 ml centrifuge tube and centrifuged at 1000r/min for 5 min,The cells were lysed with Tris-NH4Cl,3ml Tris-NH4Cl buffer resuspended precipitation,standing 3 min and plus 5ml medium,1000r/min,5min.With 2ml T cell culture medium resuspended.f).CFSE staining CFSE were 50?g powder per tube,molecular weight 557.47.Each tube plus 18ulDMSO and the suspension concentration of 5mM.Set different concentration gradients to select a better CFSE concentration.The cells were resuspended with the optimum concentration of CFSE solution.The cells were allowed to stand for 7 min at room temperature and plus 5 times the volume of serum containing the medium allowed to stand on the ice for 5 min.Washed with a large number of serum-containing medium three times,and finally resuspended with T cell culture medium.Set different concentration gradient,select the appropriate ConA concentration.The cells were incubated with mitomycin C treated tumor cells according to the need for T cells,IFN-??TNF-? and ConA made cell suspensions.g).Flow cytometryCo-culture 72h,flow cytometry detection of T cell proliferation.The cell suspension in the 12-well plate was added to the corresponding flow tube,washed once with PBS and the washed PBS was added to the corresponding tube.The cells were digested with trypsin and the cells were transferred to the corresponding flow tubes at 1000r/min,5 min,resuspended with 50 ul of 0.5%serum in PBS.Labeled antibody CD3,45min,4?.(2)Animal experimenta).Preparation of Mouse hepal-6 cell First choose a better state of murine liver cancer cells.b).Subcutaneous injection of Hepal-6 cell in miceThe cells were digested and adjusted to a concentration of 4×106cells/ml.A 6-8 week C57BL/6 mice of SPF grade were selected and each mouse was injected subcutaneously with 100 ?l of cell suspension to observe the growth of the tumor.Mice were divided into 3 groups according to tumor size(A:siNC,B:siNC?IFN-?and TNF-?,C:siB7-H1?IFN-? and TNF-?).c).Injection of small interfering RNA and cytokinesThe first day,small interfering RNA dissolved in DEPC water and about 10?g per mouse.Intratumoral injection was performed using a special in vivo transfection reagent(polyplus in vivo-jetPEI).The second day of tumor injection of cytokines TNF-? 0.5?g,IFN-? 0.05?g,small interfering and cytokines injected once every three days,small interfering and cytokine co-injection five times.d).Measurement of Tumor volumeThe tumor volume was measured on the third day and the tumor volume was measured six times throughout the procedure.Results1.IFN-? induced the expression of B7-H1 on the HCC cellsThe expression of B7-H1 mRNA in SMMC-7721 cells with different concentrations of IFN-? was different.With the increase of IFN-? concentration,the expression of B7-H1 was increased.But when IFN-? reached a certain concentration the expression of B7-H1 molecules no longer increased.Studies have found that 50ng/ml of IFN-? is the optimal concentration of B7-H1 expression.The expression of B7-H1 was increased at different concentrations of IFN-?(50ng/ml)at different times.IFN-?-induced expression of B7-H1 on the surface of HCC cells in a concentration and time-dependent manner.The results showed that IFN-? could induce the expression of B7-H1 at protein and RNA level in SMMC-7721 and HuH-7 cells.2.IFN-?-induced expression of B7-H1 on the surface of HCC cells was mainly mediated by JAK/STAT1 signaling pathway.Western-blot analysis of IFN-? induced HCC cells to express B7-H1 involved in a variety of signaling pathways.Western-blot method was used to detect the optimal inhibitory concentration.The results showed that multiple signaling pathways could affect the expression of B7-H1 induced by IFN-?,but the main effect was JAK/STAT1 signaling pathway.Transfection STAT1 and IRF1 small interference RNA and then induced by IFN-?.The results showed that the expression of B7-H1 was significantly decreased and the JAK/STAT1 signaling pathway was proved to play a major role in this process.3.The synergy of TNF-? and IFN-? in the induction of B7-H1 expression in the HCC cellsSMMC-7721,HuH-7 cells have the same phenomenon.The expression of B7-H1 was induced by TNF-?(50ng/ml),IFN-?(5ng/ml)or both.The study found that B7-H1 molecules in the RNA level and protein level were significantly increased induced by TNF-? and IFN-?.The synergy of TNF-? and IFN-y in the induction of B7-H1 expression in the HCC cells.4.The mechanism of the synergy of TNF-? and IFN-? in induction of B7-H1 expression in the HCC cellsTo further study the synergistic induction of HCC cells to express B7-H1 molecules involved in the main signal pathway.Inhibition of signal pathway activation with inhibitors and use of TNF-?,IFN-? induced cells to express B7-H1 molecules.Experiments show that the signaling pathways that play a major role in the expression of B7-H1 are also JAK/STAT1 signaling pathways.To further validate the role of the JAK/STAT1 signaling pathway.It was found that the decrease of B7-H1 after siSTAT1 and siIRFl was consistent with the effect of the inhibitors.Further detection of JAK/STAT1 signaling pathway protein levels were found to increase at the protein level.The activation of this signal pathway is enhanced.We then detected the signal pathway of the upstream molecules IFNGR1 and IFNGR2 expression.QPCR detection of TNF-? can make IFNGR1,IFNGR2 expression increased.To further study how TNF-? enhances the expression of IFNGR1 and IFNGR2.In the same manner as described above.The results showed that the expression of IFNGR1 and IFNGR2 was significantly decreased after NF-?B inhibitor and small interfering RNA.TNF-? through NF-?B signal pathway enhanced the expression of IFNGR1 and IFNGR2.5.B7-H1 expression induced by the combination of TNF-? and IFN-? have immune resistance functionThe results showed that the expression of B7-H1 was increased in Hepal-6 cells induced by IFN-? or TNF-?.The expression of Hepal-6 cells was more obvious when induced by IFN-? and TNF-?.The tumor cells of the mice were treated with TNF-? and IFN-? to express the B7-H1 cells,and then the tumor cells were co-cultured with T cells.Compared with those not treated with TNF-? and IFN-?.The results of flow cytometry showed that the average fluorescence intensity of CFSE in CD3+ cells was enhanced.The proliferation of T cells was weakened and the proliferation of T cells was inhibited.Animal experiments show,group B injected NC interference and injected TNF-?and IFN-?,group C injected B7-H1 small interfering and injected with TNF-? and IFN-?.So the expression of B7-H1 in group B was the strongest.Six tumor volume measurements showed that the tumor volume in group B was larger than that in group C and there were statistical differences.The results showed that TNF-? and IFN-?co-induced expression of B7-H1 molecules can inhibit T cells to tumor cell killing.Conclusion1.IFN-? induced the expression of B7-H1 on the HCC cells mainly through JAK/STAT1 signaling pathway2.TNF-? and IFN-? synergistically induced B7-H1 expression in the HCC cells3.TNF-? upregulated the expression of IFNGR1 and IFNGR2 through NF-?B signaling pathway,leading to the enhancement of IFN-? signaling pathway4.B7-H1 expression induced by the combination of TNF-? and IFN-? had immune resistance function... |
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