The Function And Molecular Mechanism Of GNB4 In Helicobacter Pylori-associated Gastric Cancer

BackgroundHelicobacter pylori(H.pylori)infection is a high-risk factor for the incidence of gastric cancer,defined as a class Ⅰ carcinogen by the World Health Organization,and it is involved in the pathogenesis of approximately 90%of non-cardia gastric cancer.However,the exact mechanism by which H.pylori induces gastric carcinogenesis is not fully clear at present.Aberrant DNA methylation modifications are an important mechanism in H.pylori-induced gastric carcinogenesis.The DNA methylation of malignant tumors has distinctive characteristics,and each type of tumor has tissuespecific methylation alterations of tumor-associated genes,so specific methylation genes can be used as specific biomarkers for H.pylori-associated gastric cancer,and specific methylation gene expression changes have important clinical value for early diagnosis,treatment and prognosis of gastric cancer.Studies have confirmed the involvement of G protein subunits or G proteincoupled receptors(GPCRs)in the malignant process of gastric cancer.Our preliminary bioinformatics analysis revealed that demethylation modification of the promoter region of the G protein beta subunit GNB4(Guanine nucleotide-binding protein subunit beta-4)may have an important role in H.pylori-associated gastric cancer.Moreover,GNB4 expression was significantly associated with prognosis in H.pylori-positive gastric cancer patients.However,the specific mechanism of GNB4 involvement in H.pylori infection in gastric cancer is not clear.The aim of this study was to investigate the molecular mechanism of GNB4 demethylation induced by H.pylori and to clarify the downstream regulatory network of GNB4 in gastric cancer.Objective1.To explore the role and molecular mechanisms of GNB4 demethylation modification mediating the development of H.pylori-associated gastric cancer.2.To explore the function and mechanism of GNB4 regulating the expression of Hippo-YAP1 pathway molecules involved in the malignant process of gastric cancer.Methods1.Bioinformatic approaches were used to screen the genes most related with the prognosis and DNA methylation regulation in H.pylori-associated gastric cancer.2.To analyze the correlation of GNB4 expression and clinical stage in gastric cancer using GEPIA and GEO database,and to the correlation between GNB4 and DNA methylation/demethylation enzyme molecules and Hippo pathway-related molecules expression in gastric cancer.To analyze the protein expression and prognosis of GNB4 in gastric cancer using Human Protein Atlas and Kaplan-Meier plotter database,respectively;GSEA enrichment analysis was performed to assess the relevant phenotypic pathways involved in GNB4.3.After co-culture of H.pylori(26695,SS1)with MKN45,AGS and GES1 for 6h(MOI=100),Western Blot,RT-qPCR and immunofluorescence assays combined with confocal fiber microscopy were performed to detect the effects of H.pylori infection on the expression of GNB4,TET1 and YAP1.4.CCK-8,EdU,Clone formation,and Transwell assays were used to detect the effects of aberrant GNB4 expression on the ability of H.pylori to promote proliferation,migration,and invasion of gastric cancer cells.5.Pyrophosphate sequencing and Methylation-specific PCR(MSP)assay was applied to detect the effect of H.pylori infection on the methylation level of GNB4 promoter region in cells/tissues and to identify and validate the differentially methylated CpG site region.6.We collected cancerous and para-cancerous tissues from gastric cancer patients for immunohistochemical(IHC)staining to analyze the expression of GNB4 in gastric cancer,and the correlation between GNB4 expression and the H.pylori infection and prognosis of patients.We analyzed the correlation between GNB4 expression and gastric carcinogenesis and H.pylori infection in the GEO mouse model dataset.We randomly selected H.pylori-associated patient tissues for RT-qPCR,Western Blot,and MSP assays to detect changes in GNB4 expression and DNA methylation levels.7.Using H.pylori SS1 and oncogenic agent MNU to create a gastritis model in C56BL/6J mice,hematoxylin-eosin(HE)staining was used to determine the inflammation in the stomach of mice;methylene blue staining and IHC staining were used to clarify the successful colonization of H.pylori in the stomach of mice;IHC staining was used to clarify the effect of H.pylori infection on GNB4 expression.8.We examined the regulatory relationship between TET1 and GNB4 using Western Blot assay,cell transfection with TET1 siRNA,chromatin immunoprecipitation(ChIP)and RT-qPCR assay.9.We used the constructed GNB4 overexpression gastric cancer cell line,RT-qPCR,Western Blot,YAP1 inhibitor vetiporfin(VP)intervention,and Coimmunoprecipitation(Co-IP)assay to clarify the regulatory relationship between GNB4 and YAP1.10.After the addition of VP,we used CCK-8,Transwell assay to investigate whether GNB4 exerts its cancer-promoting function through YAP1.11.We used subcutaneous tumorigenesis assay in nude mice and tail vein metastasis model in combination with VP injection to investigate the cancer-promoting function of GNB4 and whether GNB4 exerts pro-tumor growth and metastasis through YAP1.ResultsPart I:The function and mechanism of GNB4 DNA demethylation involved in H.pylori-associated gastric cancer.1.Bioinformatics analysis revealed that GNB4 is a DNA demethylation regulatory gene closely associated with the prognosis of H.pylori-associated gastric cancer.2.Compared with normal gastric tissues,GNB4 expression was increased in gastric cancer tissues;GNB4 expression was significantly correlated with the clinical stage and prognosis of gastric cancer patients.3.In vitro and in vivo experiments,the results showed that H.pylori infection significantly induced GNB4 expression;knockdown of GNB4 expression inhibited the enhancement of proliferation,migration and invasion of gastric cancer cells caused by H.pylori.4.Pyrophosphate sequencing and MSP experiments showed that H.pylori infection significantly induced DNA demethylation modifications of GNB4.5.TET1 expression was upregulated by H.pylori infection in gastric cancer cells and GES1 cells;GNB4 expression decreased and methylation level of promoter region increased after knocking down TET1 expression in gastric cancer cell lines;GNB4 expression increased after adding doxycycline to activate TET1 expression in vitro and in vivo;ChIP assay revealed that TET1 bound to the differential methylated region of GNB4 promoter.Part Ⅱ:Function and mechanism of GNB4 involved in the malignant process of gastric cancer through Hippo-YAP1 pathway-related molecules1.Online website GEPIA,RT-qPCR and Western Blot assays revealed that GNB4 was significantly correlated with Hippo pathway effector YAP1 expression but not with TAZ in gastric cancer.2.In gastric cancer cells,GNB4 overexpression activated the expression of HippoYAP1 pathway-related molecules,and overexpression of GNB4 in AGS promoted the intranuclear expression of YAP1 and its interaction with TEADs.3.In vitro and in vivo functional assays showed that VP significantly inhibited the enhanced proliferation and metastatic ability of gastric cancer cells caused by GNB4 overexpression.4.H.pylori infection significantly induced the expression of GNB4 and YAP1 in gastric cancer cells and tissues.Conclusions1.CagA-positive H.pylori upregulates the expression of DNA demethylase TET1,and TET1 could bind to the differential methylated region of GNB4 promoter,which induces DNA demethylation modification of GNB4.Then DNA demethylation modification activated GNB4 expression,which promoted the malignant process of gastric cancer.2.In H.pylori-associated gastric cancer,GNB4 initiated the expression of oncogenes downstream of the Hippo pathway by promoting the intranuclear expression of YAP1 and the binding of YAP1 to TEADs,and ultimately exerted a pro-cancer function.