Study On The Mechanism Of IL-18BP In Gastric Cancer Guided By Multi-Omics Analysis

Background and Aim:Gastric cancer is an important type of digestive system tumor that is associated with high malignancy and poor prognosis.Globally,gastric cancer ranks fifth in prevalence and is the third largest cause of cancer-related death.The development of effective diagnostic and treatment technologies for gastric cancer depends on a detailed understanding of the regulatory mechanism of gastric cancer.However,the specific mechanisms underlying the occurrence and development of gastric cancer have not been fully clarified;therefore,the molecular regulatory mechanism needs to be further explored.Bioinformatics has been widely used to study the molecular regulation of tumors.Further,we are now in the era of multi-omics analyses.Bioinformatics can be used to analyze gene expression in tumors at the RNA level through classical transcriptome sequencing(RNA sequencing [RNA-seq]),and to study the molecular regulation of tumors through proteomics,epigenomics,metabolomics,and multi-omics data.In recent years,epigenomics has become a bioinformatics research hotspot,and chromatin accessibility is an important topic in epigenomics.The most preferred method to detect chromatin accessibility is the assay for transposable accessible chromatin by highthroughput sequencing(ATAC-seq).This study aimed to identify potential key genes in gastric cancer through an integrated analysis of ATAC-seq and RNA-seq data and to study the roles and specific mechanisms of target genes in the occurrence and development of gastric cancer by analyzing clinical samples and conducting experiments using cells and animal models.Methods:1.Bioinformatics analysis: We used ATAC-seq and RNA-seq data for genetic screening.First,we identified genes with reproducible chromatin accessibility in the promoter region by analyzing ATAC-seq data from gastric cancer patients and then screened these genes to identify open chromatin regions that had strong correlations with tumor TP53 expression levels.Then,we further screened the identified genes using RNA-seq data from gastric cancer patients to find candidate genes with differential mRNA expression.Finally,we analyzed these data in combination with the prognosis and survival data of the patients with gastric cancer to identify genes related to prognosis,which were selected for the follow-up research.2.Clinical analysis: We verified the mRNA and protein expression levels of IL-18BP using real-time fluorescence quantitative PCR and immunohistochemical staining,respectively.The expression of IL-18BP in different cell types in pathological tissues was evaluated by immunohistochemical and immunofluorescence staining.The expression of IL-18BP in gastric cancer cell lines was analyzed by enzyme-linked immunosorbent assay(ELISA),and the expression of IL-18BP in different cell types in gastric cancer tissues,at different clinical stages,and in various pathological types of gastric cancer was analyzed using public databases.3.Cell and animal experiments: We used lentiviruses to construct human and mouse gastric cancer cell lines that overexpress endogenous IL-18BP.The human and mouse gastric cancer cell lines overexpressing IL-18BP were constructed in vitro through clone formation.Then,cell proliferation was analyzed using the CCK8 assay,and changes in the proliferation of the gastric cancer cells were verified in the above model.The migration ability of gastric cancer cells was assessed using both scratch and transwell cell migration assays.Using 615 strain immunocompetent mice and an MFC gastric cancer cell line from 615 strain mice,we constructed an in situ gastric tumorbearing mouse model to study the effect of IL-18BP on gastric cancer in vivo.4.Mechanism-related experiments: Using IL-18BP as a bait protein,we enriched potential IL-18BP receptor proteins in the cell membrane using a pull-down assay and then analyzed the potential IL-18BP receptor by mass spectrometry.We also verified the binding using in vitro binding experiments and evaluated the downstream pathway regulated by IL-18BP using immunofluorescence staining and western blotting.Results:1.Bioinformatics analysis results: Our bioinformatics analysis revealed 28 genes with stable chromatin accessibility in the promoter region.An integrated analysis of ATAC-seq and RNA-seq data showed that four of these candidate genes were differentially expressed,including IL-18BP,C6orf99,ASPG,and WFDC1.The results of the survival analysis showed that IL-18BP was the only gene with prognostic value.2.Clinical analysis results: The results of real-time fluorescence quantitative PCR showed that the mRNA expression of IL-18BP was significantly increased in gastric cancer.Immunohistochemical staining showed that the expression of IL-18BP protein was significantly increased in gastric cancer.We also detected IL-18BP proteinexpressing gastric cancer cells in pathological tissues.Immunofluorescence staining also showed that there were IL-18BP-positive gastric cancer cells in gastric cancer tissues.Through single cell data analysis,we found that in the gastric cancer microenvironment,IL-18BP was mainly secreted by gastric cancer cells.We also assessed the effects of interferon(IFN-γ)on IL-18BP expression in cultured gastric cancer cells using ELISA,and we found that IL-18BP protein is expressed under IFN-γ stimulation.Analysis of public databases showed that the IL-18BP expression level in patients with gastric cancer was related to the clinical TNM stage,and there was a significant increase in IL-18BP expression from T1 to T2 stage.The IL-18BP expression level in patients with gastric cancer was related to the clinicopathological classification,and IL-18BP expression was low in relatively well-differentiated pathological types,such as papillary adenocarcinoma and tubular adenocarcinoma.3.Cell and animal experimental results: We successfully constructed human and mouse gastric cancer cell lines that overexpress endogenous IL-18BP.Using clone formation and CCK8 cell proliferation assays,we found that both stimulation with exogenous IL-18BP and overexpression of endogenous IL-18BP promote the proliferation of gastric cancer cells;Using the scratch test and Transwell cell migration assay,we observed that endogenous and exogenous IL-18BP promoted the migration of gastric cancer cells.The results also showed that gastric cancer occurrence was significantly higher in the IL-18BP overexpression group.4.Mechanism-related experimental results: Protein mass spectrometry showed that CCDC25 and ILK were potential binding proteins of IL-18BP.In vitro protein binding experiments confirmed that IL-18BP could bind to CCDC25.Following stimulation with IL-18BP,ILK in the cytoplasm of gastric cancer cells migrated to the cell membrane,and phosphorylation of Ser473 of Akt and Ser9 of GSK3β occurred at1 h after IL-18BP stimulation in gastric cancer cells.These phosphorylation changes decreased at 8 h after IL-18BP stimulation.Conclusions:1.Bioinformatics: There is significant chromatin accessibility in the promoter region of IL-18BP,and IL-18BP mRNA expression is significantly upregulated in gastric cancer.Survival analysis showed that IL-18BP is a potential prognostic factor for gastric cancer.2.Clinical analysis: IL-18BP mRNA and protein levels were significantly upregulated in gastric cancer tissues when compared with the levels in adjacent tissues.Although it was known that immune cells in the tumor microenvironment can secrete IL-18BP,we reported here,for the first time,that gastric cancer cells can secrete IL-18BP.In addition,there were significant variations in the IL-18BP expression levels at different TNM stages of gastric cancer;the main difference was the significant upregulation from T1 to T2 stage.There were also differences in IL-18BP expression among the different pathological types of gastric cancer.IL-18BP expression levels were relatively low in highly differentiated pathological types,i.e.,papillary adenocarcinoma and tubular adenocarcinoma,and the prognosis of patients with high IL-18BP expression,as determined by a multi-database joint survival analysis,was relatively poor.3.Cell experiments: Treatment of cultured gastric cancer cells with IFN-γstimulated IL-18BP secretion.Stimulation with exogenous IL-18BP and overexpression of endogenous IL-18BP can promote the proliferation and migration of human and mouse gastric cancer cells.Using an immunocompetent in situ gastric tumor-bearing mouse model,we found that IL-18BP secreted by gastric cancer cells is conducive to the proliferation of gastric cancer cells in the tumor microenvironment.4.Mechanism-related experiments: IL-18BP can specifically bind to the membrane protein CCDC25 and might regulate the function of gastric cancer cells through the CCDC25-ILK-AKT/GSK3β pathway.IL-18BP stimulation can activate ILK and cause ILK in the cytoplasm to migrate to the cell membrane.IL-18BP stimulation can also activate the AKT/GSK3β signaling pathway and cause phosphorylation changes at Ser473 of AKT and Ser9 of GSK3β.