| Background Gastric cancer is one of the common malignant tumors,and it ranks second in the world in terms of tumor deaths worldwide.A large number of studies have shown that HP(Helicobacter pylori)infection promotes malignant progression and metastasis of gastric cancer,which may be related to the persistence of HP infection,resulting in local immunosuppression microenvironment,which makes gastric cancer cells escape immune surveillance.Therefore,further exploring the molecular mechanism of gastric cancer cell escape body anti-tumor immunity is the hotspot and focus of current gastric cancer research work.PD-L1(Programmed death-ligand 1)is a key member of the rapidly developing immunological checkpoint treatment in recent years.It is a receptor of PD-1(Programmed death 1)and is expressed in many types of cells.Expression of PD-L1 activates PD-L1/PD-1 signaling pathway,inhibits T cell activation and cytokine production,and escapes immune surveillance.However,the most critical stimulator regulating PD-L1 expression during anti-tumor immune response is IFN-γ.IFN-γ is a multifunctional cytokine with dual regulation.It can exert anti-tumor immune activity by inducing Th1 differentiation,CTL and dendritic cell activation;on the other hand,IFN-γ can up-regulate the expression of PD-L1 in tumor cells,so that tumor cells can escape T cell immune attack and promote tumor malignancy progress.As cytotoxic antigen protein(Cag A)is one of the most important oncogenic proteins of HP.Our preliminary results showed that transfection of Cag A into gastric epithelial GES-1 cells,simulating HP infection,reduced the expression of KLF4 in both mRNA and protein levels.KLF4 is a transcription factor that has been studied in our laboratory.Our previous experiments have found that its expression in gastric cancer cells is reduced or disappeared,which act as tumor suppressor,and participates in the immune response.It has been reported that KLF4 is a downstream target gene of IFN-γ.Therefore,we conducted preliminary experiments and found that KLF4 expression was downregulated in gastric cancer tissues,and PD-L1 was highly expressed in gastric cancer tissues.Bioinformatics analysis revealed that the PD-L1 promoter region contains two KLF4 binding sites,suggesting that there may be a negative regulatory relationship between KLF4 and PD-L1.It was found that treated with IFN-γ in gastric cancer cells up-regulated the expression of PD-L1,but the up-regulation of PD-L1 was more prominent in SK-GT5 cells with KLF4 heterozygous deletion,suggesting that KLF4 status affect IFN-γ regulates PD-L1 expression.Simultaneous transfection of different doses of Cag A also significantly up-regulated the expression of PD-L1,suggesting the effect of HP infection or Cag A transfection on PD-L1 expression.According to the preliminary results,we hypothesized that in the early stage of HP infection,the KLF4 gene functions normally,and the elevation of IFN-γ associated with HP infection can exert anti-tumor immunity by up-regulating the expression of KLF4.With the persistence of HP infection,The IFN-γ accompanying HP infection can not exert anti-tumor effect by up-regulating the expression of KLF4,but up-regulates the expression of PD-L1 to enable tumor cells to escape T cell immune attack and promote the malignant progression of gastric cancer.Whether the low expression and disappearance of KLF4 in gastric cancer cells is related to the escape of immune surveillance of gastric cancer cells,and whether the expression of KLF4 in gastric cancer cells is decreased is related to the high expression of PD-L1.Therefore,we designed experiments to verify.Research contents 1.WB detected the normal expression level of KLF4 and PD-L1 in normal gastric epithelial mucosa cells of GES-1 and three gastric cancer cells AGS,SGC-7901 and SK-GT5.2.IFN-γ treatment of AGS cells for 24 hours,detection of KLF4,PD-L1 expression.3.GES-1,AGS cells were treated with 50 ng / ml IFN-γ at 0h,2h,4h,6h,12 h,24h,and the expression of KLF4 and PD-L1 was analyzed by WB.4.50 ng/ml IFN-γ was used to treat GES-1,GES-1 KLF4 KO,AGS,and SGC-7901 cells,and the expression of KLF4 and PD-L1 was detected at protein and mRNA levels.5.GES-1,AGS cells were transfected into KLF4 Si RNA,and PD-L1 expression was detected by WB and q RT-PCR.6.GES-1 cells were transfected with KLF4 si RNA,and then treated with IFN-γ.WB,q RT-PCR was used to detect PD-L1 expression.7.SK-GT5,MGC-803 cells were transfected with exogenous KLF4 plasmid,WB was used to detect KLF4 and PD-L1 expression.8.HP and AGS were co-cultured according to 50:1,100:1,150:1 multiplicity of infection,WB was detected by KLF4,PD-L1 expression;AGS,MGC-803 cells were transfected with Cag A plasmid,WB,RT-PCR was used to detect KLF4,PD-L1 expression.9.KLF4 plasmid,STAT3 plasmid was co-transfected with PD-L1 promoter plasmid and mutant reporter plasmid,respectively.Luciferase reporter gene assay was used to detect PD-L1 reporter plasmid luciferase activity and the effect KLF4 down-regulate PD-L1.10.GES-1,AGS cells treated with 50 ng / ml IFN-γ,WB was used to detect STAT1 and STAT3 phosphorylation levels.Research results 1.KLF4 expression was higher in normal gastric epithelial GES-1,and decreased in gastric cancer cells,while PD-L1 was higher in gastric cancer cells,and KLF4 was negatively correlated with PD-L1 expression.2.IFN-γ treatment of AGS cells for 24 h found that PD-L1 expression was higher at 50 ng/ml.3.IFN-γ treatment at different time points showed that PD-L1 increased significantly at 24 h.4.GES-1 and GES-1 KLF4 KO cells treated with 50 ng/ml IFN-γ at 24 h,both KLF4 and PD-L1 expression were elevated,but PD-L1 was obviously upexpressed in GES-1 KLF4 KO cells.The same results were obtained with IFN-γ treatment of AGS and SGC-7901,suggesting a regulatory relationship between KLF4 and PD-L1.5.After GES-1 and AGS transfected KLF4 si RNA,PD-L1 was up-regulated at both protein and mRNA levels,further confirming that KLF4 negatively regulates PD-L1 expression at the transcriptional level.6.GES-1 cells were transfected with KLF4 si RNA and treated with IFN-γ.PDL1 was up-regulated at both protein and mRNA levels,further suggesting that the state of KLF4 is responsible for the regulation of PD-L1 expression by IFN-γ.7.Exogenous KLF4 was transfected into SK-GT5,and PD-L1 decreased significantly in MGC-803 cells.8.Co-culture of Cag A+ HP with different infections showed that KLF4 expression decreased gradually and PD-L1 expression gradually increased with the increase of multiplicity of infection.At the same time,Cag A plasmid was transfected into AGS and MGC-803 cells to make KLF4 Both protein and mRNA levels were down-regulated,and PD-L1 was up-regulated after Cag A transfection.9.KLF4 expression vector and PD-L1 promoter fluorescein reporter vector were co-transfected into AGS cells and found that KLF4 could significantly inhibit PD-L1 promoter activity;STAT3 expression vector and PD-L1 promoter fluorescein reporter vector were co-transfected into AGS cells to find STAT3 The PD-L1 promoter activity was significantly upregulated.Therefore,PD-L1 is a downstream target gene of KLF4,STAT3.KLF4 negatively regulates the expression of PD-L1,and STAT3 upregulates PD-L1 expression has also been reported in many literatures.10.IFN-γ treat GES-1,AGS cells increased STAT1,STAT3 phosphorylation levels,further suggest that IFN-γ can induce PD-L1 expression through STAT1 and STAT3 pathway in gastric cancer cells.Conclusions 1.HP infection/Cag A transfection leads to increased PD-L1 expression;2.KLF4 negatively regulates the expression of PD-L1 through transcriptional level;3.HP infection/Cag A transfection leads to decreased KLF4 expression,causing increased PD-L1 expression,resulting in local immunosupressive microenvironment and promoting gastric cancer cell immune escape. |
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