H.pylori Infection Induces KLF4-CXCL8 Feedback Regulation In Gastric Cancer Progression

?Background?: The incidence and mortality of gastric cancer in China are among the top five by 2015,indicating that gastric cancer is still a serious factor affecting people's life and health.Up to now,the immunotherapy of gastric cancer has developed rapidly and has achieved enormous clinical treatment effects,but it still has not achieved the expected results.There may be other factors that interfere with immunotherapy.Helicobacter pylori(H.pylori)infection is a key factor in the occurrence and development of gastric cancer.Among them,the cytotoxin-associated antigen(CagA)protein is the main carcinogen,and H.pylori enters cells through a specific type IV secretion system.Studies have shown that CagA can inhibitor the expression of KLF4 in gastric cancer,and the mechanism studies have shown that CagA can silence or inactivate KLF4 expression through methylation of the KLF4 promoter region and MIR155 pathway.KLF4 is a zinc factor-containing transcription factor.Studies have shown that KLF4 acts as a tumor suppressor gene in gastric cancer,and it has been confirmed that KLF4 inactivation is important to gastric cancer treatment.Our team mainly studied the mechanism of CagA leading to the inactivation of KLF4.Then,what is the specific relationship between the low expression or loss of tumor suppressor protein KLF4 and the promotion of gastric cancer development? CXCL8 is a cytokine of the chemokine family,and binds to specific receptors and participates in a variety of inflammatory responses.It is mainly secreted by monocytes and macrophages.Studies have shown that CXCL8 is the only gene that is most significantly up-regulated in the whole genome of H.pylori infect gastric mucosal epithelial cells 24 hours later.CXCL8 is a very important inflammatory chemokine induced by H.pylori infection.In gastric cancer,CXCL8 plays a biological role in combination with its receptors CXCR1 and CXCR2.Some studies have found that CXCL8 can chemo bone marrow-derived suppressor cells(MDSCs)by binding to CXCR2 receptors on the cell surface to reach the tumor site and expand,and MDSCs can inhibit the activation of T cells through multiple mechanisms.In response,an antitumor immune response is generated.The use of anti-CXCR2 monoclonal antibodies to block MDSCs from chemotaxis to the tumor area significantly enhanced the anti-PD-1 immune checkpoint blocking treatment effect.Therefore,how to effectively inhibit and block the high expression of CXCL8 in gastric cancer and break the local poor immunosuppression microenvironment is an important to improve the effect of comprehensive immune treatment of gastric cancer.Bioinformatics analysis found that the promoter region of the CXCL8 gene immediately adjacent to the TATA box contains two potential binding sites for KLF4.Pre-experimental results showed that the KLF4 expression vector co-transfected with the CXCL8 gene promoter luciferase reporter gene could strongly inhibit the gene promoter luciferin active.Based on this,whether H.pylori infection caused the upregulation of the inflammatory factor CXCL8 is related to the down-regulation of KLF4,and continuous H.pylori infection in gastric cancer caused KLF4 down-regulation to further cause CXCL8 up-regulation,thereby inducing the local immune suppressive microenvironment of the tumor and promoting the progression of gastric cancer.Design the following experimental methods to test the hypothesis.?Research contents?: 1.Tissue chips were used to detect the expression of CXCL8 and KLF4 in the para-cancerous and cancerous tissues of gastric cancer patients,and statistical analysis was performed.2.GES-1,AGS,MGC80 cells were transfected with CagA,and CXCL8 RNA and protein expression was detected by q RT-PCR and ELISA after 48 h.3.GES-1,AGS,MGC80 cells were co-cultured with H.pylori according to multiple infection at 25: 1,50: 1,100: 1(MOI: H.pylori: cell),and the expression of CXCL8 was detected by q RT-PCR and ELISA after 48 h.4.AGS,MGC-803 cells were transfected with different doses of KLF4,and the expression of CXCL8 was detected by q RT-PCR and ELISA after 48 h.5.GES-1 and AGS cells were transfected with KLF4-Si RNA,the expression of CXCL8 was detected by q RT-PCR and ELISA.6.Construct the CXCL8 promoter reporter plasmid(WT)and its mutant reporter plasmid(Site1,Site2,Double site).The constructed plasmids were cotransfected with KLF4 expression vector plasmids and mini-tk plasmids,and the luciferase activity of the CXCL8 promoter reporter plasmid was detected using a dual luciferase reporter gene kit.7.The CXCL8 promoter reporter gene plasmid was co-transfected with the CagA plasmid and mini-TK plasmid,and the double luciferase reporter gene kit was used to detect the luciferase activity of the CXCL8 promoter reporter plasmid.8.The CXCL8 promoter reporter gene plasmid is co-transfected with the KLF4 plasmid,CagA plasmid,and mini-TK plasmid.The dual luciferase reporter gene kit detects the luciferase activity of the CXCL8 promoter reporter plasmid,and see if KLF4 can reverse the regulation of CagA on CXCL8.It also see whether CagA can reverse the regulation effect of KLF4 on CXCL8.9.CHIP assay to detect whether KLF4 binds to the CXCL8 promoter region.10.Recombinant human CXCL8 protein was used to stimulate GES-1/AGS cells with 6 h and 15 days,and KLF4 expression were detected by WB.The proper concentration(3ng/m L)was chosen to stimulate GES-1/AGS cells for 15 days,and detect the capacity of cells proliferation and migration.11.AGS and MGC803 were separately stimulated with CXCL8 recombinant protein,and another grow co-transfected with KLF4 expression vector to detect cell proliferation and migration ability.12.Construct MGC803 cell chronically treated with CXCL8 recombinant protein for 30 days,then test their malignant behavior and proliferation ability.Performing subcutaneous tumor formation experiments using BALB / c nude mice to verify the effect of CXCL8 on tumor growth in vivo and KLF4 expression.?Research results?: 1.Tissue chip test found that the expression of CXCL8 and KLF4 in gastric cancer patients and adjacent tissues was negatively correlated,and had statistical significance.2.GES-1,AGS,MGC803 cells were transfected with CagA,and the expression of CXCL8 at both RNA and protein levels was increased by q RTPCR and ELISA at 48 h.3.GES-1,AGS cells and MGC-803 cells were co-cultured with H.pylori at 25: 1,50: 1,100: 1 infection multiple numbers.After 48 h,q RT-PCR and ELISA detected CXCL8 protein and RNA expression levels were up-regulated.4.After the AGS and MGC-803 cells were transfected with different doses of KLF4,q RT-PCR and ELISA were used to detect CXCL8 down-regulation in both RNA and protein levels after 48 h.5.After the transfection of KLF4-Si RNA with GES-1 and AGS cells,the expression of CXCL8 protein and RNA was significantly up-regulated by q RTPCR and ELISA.6.When CXCL8 promoter reporter gene plasmid and KLF4 plasmid were cotransfected into GES-1 cells,it was found that KLF4 could significantly inhibit the activity of CXCL8 promoter,and as the dose of KLF4 increased,its inhibition of CXCL8 was more obvious,while CXCL8 promoter When the mutant reporter gene plasmid and KLF4 plasmid were co-transfected into GES-1 cells,the inhibitory effect of KLF4 on CXCL8 disappeared.The same results were obtained in AGS cells.7.When CXCL8 promoter reporter gene plasmid and CagA plasmid were cotransfected into GES-1 / AGS cells,CagA was found to significantly upregulate CXCL8 promoter activity.8.When CXCL8 promoter reporter gene plasmid was cotransfected with KLF4 plasmid and CagA plasmid at the same time in GES-1 cells,it was found that when CXCL8 and KLF4 doses were constant,CagA could reverse the negative regulation effect of KLF4 on CXCL8 when the dose of CagA increased.The same results were obtained in AGS cells.When the doses of CXCL8 and CagA are constant,with the increase of KLF4 dose,KLF4 can reverse the upregulation effect of CagA on CXCL8.The same results were obtained in GES-1 cells.9.HA-KLF4 plasmid was transfected in AGS cell.Compared with the control group,the expression of CXCL8 in the HA-KLF4 plasmid group was significantly enhanced compared with the control group.10.After treating GES-1 and AGS cells with CXCL8 recombinant human protein at different concentrations,KLF4 expression decreased regardless of the length of stimulation.A concentration of 3 ng/m L was chosen to stimulate GES-1/AGS cells for 15 days,the results show promotes cells proliferation,colony formation and migration.11.AGS,MGC803 stimulated with CXCL8 recombinant protein,MTT,transwell,and plate clone experiments showed that the cell proliferation and migration ability were enhanced.But KLF4 expression vector was transfected at the same time with CXCL8 protein treatment,the cell proliferation and migration capacity is significantly reduced.12.The constructed MGC803-CXCL8 was treated chronically with CXCL8 recombinant protein for 30 days,and its malignant behavior and proliferation ability were enhanced.In vivo nude mice tumor formation experiments showed that the tumors in the CXCL8 treatment group had faster growth.Tumor tissue immunohistochemical results showed that KLF4 protein expression decreased,while Ki67 protein expression increased.?Conclusions?: 1.CXCL8 is negatively correlated with KLF4 expression in gastric cancer tissues.2.H.pylori infection or CagA gene transfection up-regulated the expression of CXCL8.3.Increasing KLF4 expression can inhibit CXCL8 expression,while KLF4-Si RNA transfection reversly up-regulate CXCL8 expression.4.Dual luciferase reporter gene assay show that KLF4 negatively regulates CXCL8 expression at the transcription level.5.Chromatin immunoprecipitation experiments showed that KLF4 can bind to the CXCL8 promoter region.6.In vivo and in vitro experiments verify that CXCL8 inhibit KLF4 expression,and promote cell proliferation,migration,and tumor growth.