The Mechanisms Of PD-L1 Expression Induced By Tumor Inflammation Microenvironment In Hepatocellular Carcinoma Cells

BackgroundsNowadays,the incidence of Hepatocellular Carcinoma Cells(HCC)ranks second in the world,just behinds lung cancer.HCC seriously affects human health with its high incidence,easy metastasis and easy recurrence.The etiology and exact molecular mechanism of primary hepatocellular carcinoma are not fully understood.The pathogenesis of HCC is a multifactorial,multistep complex process,which is influenced by environmental and genetic factors.In recent years,tumor immunotherapy has received considerable attention during the medical field.Immunotherapy includes two types:adoptive cellular immunotherapy and check-point blockade.Check-point blockade includes CILA-4 monoclonal antibodies and PD-1/PD-L1 monoclonal antibodies.PD-L1,also known as B7H1,is highly expressed in various solid tumor tissues and is negatively correlated with the overall survival of the cancer patients.PD-L1 could inhibit anti-tumor immunity through various function,and the main function of that is to induce apoptosis or anergy of CTL cells when engaged with the PD-1,so the effect T cells can not produce cytokines and cytotoxic substances,finally resulting in tumor immune escape of tumor and its progression.Tumor microenvironment is a complex system.It consists of different stromal cells and plays an important role in the development of tumor.Tumor-associated macrophages(TAM)exist in the interstitium of the tumor.Peripheral blood mononuclear cells migrate to tumor tissue and further develop into tumor-associated macrophages with the assistance of different chemokines and cytokines.Do the TAM affect the expression of PD-L1 molecules in HCC?Which type of macrophage can affect it?What is the function of this role?The explainations of those questions are the main purposes of the first part of this study.In the tumor microenvironment,apart from a lot of different stromal cells(such as TAM,T cells,MDSCs etc.),there are also various cytokines,including IFN-?,TNF-? and TGF-?,which have played an important role during tumor progression.The "adaptive resistance hypothesis" of tumor immune escape proposed by Lieping Chen pointed out that T cell infiltrate around tumor and secrete IFN-?,IFN-? can induce high expression of PD-L1 in tumor cells,resulting in adaptive resistance of tumors.Some studies have shown that c-Myc can regulate the expression of PD-L1 in lymphoma.Does c-Myc can regulate the expression of PD-L1 induced by IFN-? in HCC?How does this process be adjusted?Those are the second part of our study.ObjectivesConcerning above problems,we divided the subject into two parts.In the first part,We detected the effect of TAM on PD-L1 in HCC cells and analysed the mechanisms;In the second part,we used IFN-y to induce high expression of PD-L1 in HCC cells and detected whether c-Myc had an effect on the expression of PD-L1,and explored the mechanisms.Methods and results?:The effect of TAM on the expression of PD-L1 in HCC(?)The supernatant of Ml-like macrophages up-regulate the expression of PD-L1 in HCC1.Induction and identification of M1-like and M2-like macrophagesTHP-1 cells were treated with PMA to be adhered to the plate,then the cells were induced to M1-like macrophages and M2-like macrophages through using LPS and IL-4 respectively.After harvesting cells,RNA was extracted and cDNA was obtained by reverse transcription.The phenotype of two types of macrophages was identified by PCR.M1-like macrophages express high level of TNF-a and IL-6,while M2-like macrophages express high level of CD206.2.The supernatant of M1-like macrophages up-regulate the expression of PD-L1 at mRNA level and protein levelM1 and M2 like macrophages were cultured for 48 hours to obtain the M1-like macrophages supernatant(M1S)and M2-like macrophages supematant(M2S).BEL-7402 and Huh-7 cells were treated with M1S and M2S for 6h.RNA was extracted and cDNA was obtained by reverse transcription,the expression of PD-L1 at mRNA level was detected by RT-PCR.BEL-7402 and Huh-7 cells were stimulated by the above two supernatants for 24 hours.The cells were harvested and the expression of PD-L1 was detected by flow cytometry at the protein level.The results showed that M1S induce high expression of PD-L1 at both mRNA and protein levels.3.The high expression of PD-L1 at mRNA level induced by M1S is time dependentTo confirm that whether MIS could induce the high expression of PD-L1,Huh-7 cells were treated with M1S for 4h,6h and 24h.Then the expression of PD-L1 was detected by RT-PCR.The results showed 6h is the optimal time point for the effect of M1S on the expression of PD-L1.(?)The mechanisms of high expression of PD-L1 induced by M1S in HCC1.Cytokines that could affect the expression of PD-L1 in M1S1)Firstly,we detected the content of IL-1? in the supernatant of macrophages.The concentration of IL-1? in M1S and M2S were detected by human IL-? Elisa kit.Huh-7 cells were pretreated with IL-1 inhibitor IL1RA(200ng/ml and 500ng/ml)for 30min and then were treated with MIS for 6h.The expression of PD-L1 at mRNA level was detected by RT-PCR.In order to further prove the effect of IL-1? on the expression of PD-L1,BEL-7402,Huh-7 cell lines were transfected with siIL-1R1 for 48h,then were treated with MIS for 6h,cells were harvested and cDNA were obtained by reverse transcription,the expression of PD-L1 was detected by RT-PCR.HCC cell lines were treated with M1S for 24h,Then the expression of PD-L1 was detected by flow cytometry at protein level.The results showed that the content of IL-1? in Ml supernatant was 500pg/?l,the content of IL-1? in M2 supernatant was 180pg/?l.After using IL1RA and siIL-1R1,the expression of PD-L1 in HCC cells induced by M1S was considerably inhibited.2)BEL-7402 and Huh-7 cell were transfected with the siIFNAR2 and siIFNGR1 respectively.After 48 hours,cells were treated with M1S for 6h and RT-PCR was used to detect PD-L1 expression,The results showed that the upregulation effect of MIS on expression of PD-L1 was not affected by the two types of siRNA.Those results suggested that the two types interferon did not play a significant role in this process.3)BEL-7402 and SMMC-7721 were transfected with the siTNFR for 48h and were treated with MIS for 6h or 24h and RT-PCR was used to detect PD-L1 expression at mRNA level.Flow cytometry was used to detect PD-L1 expression at protein level.The results showed that the upregulation effect of MIS on PD-L1 expression was obviously inhibited by siTNFR.4)SMMC-7721 and Huh-7 were transfected with siIL-6R for 48h and were treated with MIS for 6h or 24h,RT-PCR was used to detect PD-L1 expression at mRNA level.Flow cytometry was used to detect PD-L1 expression at protein level.The results showed that IL-6 also plays an important role in this process.5)We used IL-6(50ng/ml)alone,TNF-?(50ng/ml)alone,IL-1?(50ng/ml)alone,or the combination of the three cytokines(MIX)to stimulate BEL-7402 or Huh-7 for 6h or 24h respectively,the expression of PD-L1 was detected at mRNA level and protein level.The results showed that all of the three cytokines increase the expression of PD-L1 in HCC,and the MIX induce higher PD-L1 expression in HCC than the individual cytokines.2.Transcriptional factors that could affect the regulation of PD-L1 expression induced by MISThrough the above experiments,we identified that IL-6,TNF-?,IL1-? play an important role in the process.Some studies have proven that TNF-? and IL-1? could induce the expression of IRF1.TNF-? could activate NF-kB(P65)pathway while IL-6 could activate JAK-STAT3 signaling pathway,hence we decided to investigate if transcriptional factors IRF1,NF-?B(P65)and STAT3 could play an important role in the expression of PD-L1 induced by M1S.1)We synthesized siIRF1,siP65 and siSTAT3 respectively,then transfected them into BEL-7402 or Huh-7 cells.After 48 hours,cells were treated with M1S for 6 hours.The expression of PD-L1 in HCC cells was detected by RT-PCR at mRNA level.The results showed that the upregulated effect of MIS on PD-L1 expression was significantly inhibited by the three transcriptional factors siRNA.2)In order to determine if P65,IRF1 and STAT3 were directly acted on PD-L1 promoter region after treated with M1S,we carried out the Chip experiments.Chip experimental results showed that with treatment of M1S,the activity of these three transcription factors to PD-L1 promoter was enhanced,to confirm if the three transcriptional factors are binding to PD-L1 promoter directly or indirectly,we carried out following experiments.The possible binding sites of p65 on PD-L1 promoter are nt-607to-597 and nt-62to-53;the possible binding sites of IRF1 on PD-L1 promoter are nt-159to-148 and nt-109to-97,the possible binding sites of STAT3 on PD-L1 promoter are nt-481to-470 and nt-134to-124.The mutated plasmids were transfected into BEL-7402 cells,then were treated with MIS for 6h.Luciferase reporter gene assay was used to detect the activity of PD-L1 promoter.The experimental results showed that P65 and STAT3 might not directy bind to the PD-L1 promoter.IRF1 was directly bound to the PD-L1 promoter region at the site(nt-159to-148).3.Effect of M1S on chromatin histone modification on PD-L1 promoterWe designed Chip primers for two sites of PD-L1 promoter at nt-739to-597 and nt+416to+581.We used Chip grade antibody anti-H3K9Ac and anti-H3K4Me to carry out the test in the BEL-7402 cell lines,and observed the changes of chromatin histone modification after treatment of M1S.The results showed that the MIS had no effect on chromatin histone modification on PD-L1 promoter.?:The regulation and mechanisms of c-Myc on the IFN-? induced PD-L1 expression(?):Detection of c-Myc expression in different cell linesFive cell lines SMMC-7721,Huh-7,BEL-7402,HepG2 and Hep3B were selected to detect the expression level of c-Myc at mRNA level and protein level by RT-PCR and WesternBlot.(?):The effect of c-Myc on the expression of PD-L1 induced by IFN-?HCC cell lines(SMMC-7721,BEL-7402,Hep3B)with high expression of c-Myc were selected and transfected with sic-Myc for 48h,then were treated with IFN-?(50ng/ml)for 24h.The expression of PD-L1 was detected by flow cytometry at protein level.Results showed that the expression of PD-L1 was increased with transfection of sic-Myc.(?):The mechanisms of c-Myc inhibit the expression of PD-L1 induced by IFN-?1.The effect of c-Myc on the expression of PD-L1 at transcriptional levelSMMC-7721,BEL-7402 and Hep3B were transfected with sic-Myc for 48h,then were treated with IFN-?(50ng/ml)for 6h.The expression of PD-L1 was detected by RT-PCR at mRNA level.We transfected BEL-7402 cell lines with sic-Myc for 24h,then cotransfected the mixture of luciferase reporter plasmid PGL3-PD-L1 promoter and pRL-TK-Renilla lucif-erase plasmid,luciferase assay showed sic-Myc could enhance activity of PD-L1 promoter.In addition,BEL-7402 and SMMC-7721 cell lines were transfected with sic-Myc and pRK5-c-Myc plasmids,then were stimulated with IFN-? for 6h.Results showed that pRK5-c-Myc plasmid could inhibit PD-L1 expression.2.The effect of sic-Myc on the IFN-y signaling pathwayHCC cells were transfected with sic-Myc for 48h and were stimulated with IFN-? for 5min or 15min.The expression of total STAT1(T-STAT1)and p-STAT1 were detected by RT-PCR and WesternBlot.The results showed that sic-Myc could increase the expression of T-STAT1 and p-STAT1.Conclusions?:The effect of TAM to the expression of PD-L11.The supernatant secreted by M1-like macrophages can up-regulate the expression of PD-L1 in HCC cells;2.TNF-?,IL-6,IL-1? secreted by M1-like macrophages are responsible for the upregulation of PD-L1 in HCC cell lines;3.P65?IRF1?STAT3 play an important role in the expression of PD-L1 induced by MIS;4.M1S could not affect H3K9 acetylation and H3K4 methylation of PD-L1 promoter in HCC.?:The regulation and mechanisms of c-Myc on the expression of IFN-? induced PD-L11.c-Myc could inhibit the expression of PD-LI induced by IFN-y;2.c-Myc could inhibit the expression of PD-L1 through inhibiting T-STAT1 and p-STAT1.