| Objective: To evaluate the pro-cancer risk and mechanism of Epstein-Barr virus(EBV)in the development of gastric cancer(GC).Methods: Firstly,the study used clinical data to describe the distribution characteristics of plasma EBV DNA in the subjects with non-atrophic gastritis(NAG),atrophic gastritis(AG),and GC.We explored the risk of EBV promoting the occurrence of AG and GC.Secondly,TCGA and GEO databases were used to analyze the expression pattern of m6 A methylation regulators between EBV-associated gastric cancer and EBV-negative gastric cancer.We constructed a prediction model for EBV-associated gastric cancer.We identified GO and KEGG functional enrichment of m6A-related differentially expressed genes(DEGs)in EBV-associated gastric cancer.We quantified the release level of immune cells and inflammatory factors in the tumor microenvironment(TME).Finally,the CCK-8 cell proliferation test,Transwell test,and flow cytometry were used to verify the influence of IGFBP1 on the proliferation,migration,and apoptosis of EBV-positive gastric cancer cell lines.Results: A total of 884 patients who underwent digestive endoscopy in the Digestive Endoscopy Center of Tongji Hospital,Tongji Medical College of Huazhong University of Science & Technology were enrolled in the study.The subjects were classified as 353(39.9%)NAG patients,284(32.1%)AG patients,and 247(27.9%)GC patients.Plasma EBV DNA positive could increase the risk of AG and GC by 1.438-fold(P=0.030)and 1.478-fold(P=0.024),respectively.Plasma EBV DNA positive significantly increased the risk of GC in young,male,and H.pylori-negative patients(P<0.05).In addition,EBV DNA positive could also promote the occurrence of AG in H.pylori-negative patients(P=0.011).There was no statistical correlation between EBV DNA and H.pylori infection status(P=0.772).m6 A methylation regulators were involved in the occurrence and development of EBV-associated gastric cancer.Compared with EBV-negative gastric cancer,the expression levels of m6 A methylation regulators WTAP,RBM15 B,CBLL1,LRPPRC,HNRNPA2B1,IGFBP1,and IGF2BP1 were significantly down-regulated in EBVassociated gastric cancer(P<0.05).The overall survival rate of EBV-associated gastric cancer patients with a lower expression level of IGFBP1 was significantly higher(P=0.046).The nomogram model based on the IGF2BP1 and IGFBP1 might be beneficial to the screening,diagnosis,and treatment of EBV-associated gastric cancer.Immunity pathways of EBV-associated gastric cancer were significantly activated,rich in immune cells and inflammatory factors infiltration.The expression level of IGFBP1 was significantly lower in EBV-positive gastric cancer cell lines than that in EBV-negative gastric cancer cell lines(P<0.05).IGFBP1 overexpression significantly attenuated the proliferation and migration,and promoted the apoptosis of EBV-positive gastric cancer cell lines.Interfering IGFBP1 significantly promoted the proliferation and migration,and attenuated the apoptosis of EBVnegative gastric cancer cell lines.Conclusions: EBV played a pro-tumor role in the occurrence and development of gastric cancer,especially in young,male,and H.pylori-negative individuals.m6 A methylation regulators influenced the expression level of immune cells and inflammatory factors in EBV-associated gastric cancer,which could remodel TME that was classified as an immuneinflamed phenotype.EBV-associated gastric cancer was referred to as a “hot” tumor.IGFBP1 affected the proliferation,migration,and apoptosis of EBV-associated gastric cancer.Part Ⅰ: The Role of Epstein-Barr Virus in the Development of Gastric CancerObjective: To describe the distribution characteristics of plasma EBV in patients with NAG,AG,and GC,and evaluate its risk in promoting the occurrence of AG and GC.Methods: The study retrospectively enrolled 884 patients who underwent upper digestive endoscopy in the Digestive Endoscopy Center of Tongji Hospital,Tongji Medical College of Huazhong University of Science & Technology.The symptoms included epigastric pain,abdominal distension,regurgitation,heartburn,etc.Firstly,the subjects were divided into NAG,AG,and GC groups.We analyzed the clinical characteristics of the three groups.Secondly,the subjects were divided into EBV DNA positive group and EBV DNA negative group according to the detection of plasma EBV DNA.We analyzed the distribution characteristics of plasma EBV DNA of the subjects and the risk of promoting AG and GC.Then,we analyzed the relationship between EBV DNA and H.pylori.We explored the role of co-infection in promoting cancer.Finally,we used the multinomial logistic regression analysis to explore the risk factors of AG and GC.Results: A total of 884 patients with a median age of 53.8 ± 9.5 years(27~86 years)were enrolled in the study.The subjects were classified as 353(39.9%)NAG patients,284(32.1%)AG patients,and 247(27.9%)GC patients.There was correlation between gastric mucosal lesions and age,BMI,H.pylori,and EBV DNA(P<0.05).Plasma EBV DNA was correlated with gender and gastric mucosal lesions(P<0.05).Compared with NAG patients,plasma EBV DNA positive could increase the risk of AG and GC by 1.438-fold(P=0.030)and 1.478-fold(P=0.024),respectively.In young patients(<60 years old),plasma EBV DNA positive could increase the risk of GC by 1.902-fold(P=0.005).In male patients,plasma EBV DNA positive could increase the risk of GC by 1.879-fold(P=0.002).In H.pylorinegative patients,plasma EBV DNA positive could increase the risk of AG and GC by 1.684-fold(P=0.011)and 1.629-fold(P=0.020).Regardless of gastric mucosal atrophy or cancer,there was no correlation between EBV DNA and H.pylori infection status(P>0.05).Age ≥ 60,H.pylori positive,and EBV DNA positive were high-risk factors for the development of AG and GC(P<0.05).Conclusions: Plasma EBV DNA positive was a risk factor for the occurrence and development of AG and GC,which increased the risk of AG and GC,especially in young,male,and H.pylori-negative individuals.Part Ⅱ: The Potential Role of m6 A Methylation Regulation in EBV-associated Gastric CancerObjective: To explore the expression pattern and potential mechanism of m6 A methylation regulators in EBV-associated gastric cancer.Methods: The study was carried out by using bioinformatics analysis methods.Firstly,we analyzed the expression pattern of m6 A methylation regulators and the clinical prognosis between EBV-associated gastric cancer and EBV-negative gastric cancer.Secondly,random forest(RF)and support vector machine(SVM)models were constructed.We selected the best training model to predict the occurrence of EBV-associated gastric cancer.We constructed a nomogram model to predict the occurrence of EBV-associated gastric cancer.Then,we screened m6A-related DEGs in EBV-associated gastric cancer.GO and KEGG pathway enrichment analysis were performed to analyze the functions and signal pathways related to DEGs.Finally,we quantified the relative abundance of immune cells and inflammatory factors in TME.Results: Compared with EBV-negative gastric cancer,the expression levels of m6 A methylation regulators WTAP,RBM15 B,CBLL1,LRPPRC,HNRNPA2B1,IGFBP1,and IGF2BP1 were significantly down-regulated(P<0.05).The lower expression of IGFBP1 indicated that the overall survival rate of GC patients is significantly higher and the prognosis is better(P=0.046).RF model had higher accuracy than the SVM model and might be used as the best model to predict the occurrence of EBV-associated gastric cancer.According to the mean decrease Gini index based on the RF model,IGF2BP1 and IGFBP1 played an important factor in the occurrence and development of EBV-associated gastric cancer.The nomogram model based on the IGF2BP1 and IGFBP1 might be beneficial to the screening,diagnosis,and treatment of EBV-associated gastric cancer.GO and KEGG functional enrichment analyses showed that the immunity pathways were significantly activated and rich in immune cell infiltration in EBV-associated gastric cancer.Compared with EBV-negative gastric cancer,the infiltration of activated CD4+ T cells,activated CD8+ T cells,monocytes,activated dendritic cells,and plasmacytoid dendritic cells were significantly up-regulated in EBV-associated gastric cancer(P<0.001).In EBV-associated gastric cancer,the expression level of proinflammatory factors IL-17,IL-21,and IFN-γ and immunosuppressive factor IL-10 were significantly increased(P<0.05).Conclusions: The expression levels of m6 A methylation regulations were different between EBV-associated gastric cancer and EBV-negative gastric cancer,which affected the clinical prognosis.m6 A methylation regulated EBV-associated gastric cancer to remodel immuneinflammatory TME.EBV-associated gastric cancer was referred to as a “hot” tumor.Part Ⅲ: The Regulatory Role of IGFBP1 in EBV-associated Gastric CancerObjective: To verify the expression level of IGFBP1 in EBV-negative gastric cancer cell lines and EBV-positive gastric cancer cell lines and its influence on biological functions.Methods: Western blot was used to analyze the expression level of IGFBP1 in EBVnegative gastric cancer cell lines and EBV-positive gastric cancer cell lines.Plasmid overexpression and RNA interference technology were used in gastric cell lines.CCK-8 cell proliferation test,Transwell test,and flow cytometry were used to evaluate the influence of IGFBP1 on the proliferation,migration,and apoptosis of EBV-positive gastric cancer cell lines.Results: Western blot showed that the expression of IGFBP1 in EBV-positive gastric cancer cell lines was significantly lower than that in EBV-negative gastric cancer cell lines(P<0.05).IGFBP1 overexpression significantly attenuated the proliferation and migration,and promoted the apoptosis of EBV-positive gastric cancer cell lines.Interfering IGFBP1 significantly promoted the proliferation and migration,and attenuated the apoptosis of EBVnegative gastric cancer cell lines.Conclusions: IGFBP1 could attenuate the proliferation and migration,and promote the apoptosis of EBV-positive gastric cancer cell lines... |
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