The Role And Molecular Mechanism Of RASAL2 In Helicobacter Pylori-induced Gastric Tumorigenesis

Objective: Gastric cancer is still a serious public health problem worldwide.In recent years,its high morbidity and mortality have remained in Asia,especially in China,Japan and South Korea,of which the mortality of gastric cancer often ranks among the forefront of malignant tumors.Although the innovation of gastroscopy detection technology has increased the diagnosis rate of early gastric cancer,most patients have developed advanced gastric cancer when they are diagnosed.At present,comprehensive treatment with surgery supplemented by postoperative chemotherapy has very limited benefits for further improving the survival of patients,and it is urgent to explore new molecular targets.The strongest known pathogenic factor for gastric cancer is Helicobacter pylori(H.pylori),which is defined as a Class I carcinogen by the World Health Organization and participates in the pathogenic process of about 90%of gastric non-cardiac cancer.More and more studies have shown that Helicobacter pylori promotes the occurrence and development of gastric cancer by activating tumor-promoting genes and(or)suppressing tumor suppressor genes through genetic and epigenetic modifications.H.pylori can also directly or indirectly activate transcription factors,such as NF-κB,STAT3 and β-catenin,to further regulate the expression of downstream genes,and play an important role in the formation,metastasis,drug resistance and maintenance of stem cell characteristics of gastric cancer.RAS protein activator like 2(RASAL2)belongs to the RAS GTPase activator protein family and plays important and different roles in a variety of malignant tumors,but its role and molecular mechanism in gastric cancer have not been elucidated.This study aims to clarify the role of H.pyloriinduced RASAL2 in the formation of gastric cancer and its molecular mechanism,and provide more theoretical basis for the development of new treatment strategies for gastric cancer.Methods:1.Thirty-six cases of human gastric cancer and its adjacent tissues were collected and high-throughput RNA sequencing was performed.Another 10 cases of Tff1 knockout mice(5 cases with H.pylori infection and 5 cases without infection)were collected and high-throughput RNA sequencing was performed.Analysis the significant difference genes in each data set and intersection to obtaining the key gene RASAL2 that might regulate H.pylori-induced carcinogenesis.And verify its expression through TCGA and GEO public data sets.2.Use the model of H.pylori infection and Chromatin immunoprecipitation(Ch IP)and luciferase assayin vitro to verify that H.pylori transcriptionally regulatethe expression of RASAL2.3.Using GO pathway enrichment analysis to predict the biological role of RASAL2 in gastric cancer,and use in vitro models(sphere forming experiments and organoid experiments)and in vivo experiments to verify its biological functions.4.Using gene set enrichment analysis(GSEA)to predict the downstream signaling pathway of RASAL2,and verifying the downstream regulatory mechanism of RASAL2 through q RT-PCR and Western Blotting combined with in vitro H.pylori infection model.5.Collecting 365 cases of gastric cancer tissue microarrays in our center,using immunohistochemistry to detect RASAL2 expression,and analysis its effect on chemotherapy sensitivity and prognosis.Results: 1.Analysis of our center and public data sets showed that the expression level of RASAL2 in gastric cancer tissues was significantly higher than that in adjacent tissues(P<0.05).2.In vitro H.pylori infection model showed that H.pylori infection up-regulated RASAL2 m RNA and protein expression.Treatment with TNFα(or overexpression of p65)promoted,while BAY 11-7082 inhibited RASAL2 m RNA and protein expression.Ch IP results showed that NF-κB directly binds to the RASAL2 promoter region.Luciferase experiments showed that NF-κB activated the transcriptional activity of the RASAL2 promoter.3.After RASAL2 was stably silenced in MNK28 and MNK45 cell lines,the diameter of spheres(P<0.01)and the number of spheres formed significantly decreased(P<0.05).After RASAL2 was stably silenced in organoids derived from human gastric cancer,the diameter(P<0.01)and number(P<0.05)of the organoids were significantly down-regulated.The nude mouse subcutaneous tumorigenesis model further confirmed that silencing RASAL2 significantly inhibited the ability of subcutaneous tumor formation(P<0.001).4.By using gene silencing and transient overexpression,we found that RASAL2 upregulated β-catenin transcription activity in an AKT/GSK-3β-dependent manner,and RASAL2 depletion or AKT inhibitors eliminated H.pylori-inducedβ-catenin phosphorylationand nuclear translocation.In order to study the underlying mechanism,we revealed that RASAL2 binds protein phosphatase 2A(PP2A),which inhibits PP2 A kinase activity and further activates the AKT/GSK-3β/β-catenin axis.5.Immunohistochemistry results from tumor microarrays showed that the positive expression rate of RASAL2 protein in gastric cancer tissues was significantly higher than that of adjacent tissues.Multivariate analysis showed that the patient’s age,tumor location,AJCC stage and RASAL2 expression were independent prognostic factors for the overall survival rate of gastric cancer.Clinicopathological analysis showed that the abnormal overexpression of RASAL2 in human gastric tumors was related to poor prognosis and adjuvant chemotherapy resistance.Conclusions: 1.Abnormal overexpression of RASAL2 is closely related to poor prognosis and adjuvant chemotherapy resistance,and is expected to be a new target for predicting the prognosis and chemotherapy sensitivity of gastric cancer.2.H.pylori-induced NF-κB directly binds to the RASAL2 promoter region through NF-κB and transcriptionally regulates the expression of RASAL2.3.RASAL2 plays an important role in promoting gastric tumorigenesis.4.RASAL2 binds to and inhibits PP2 A kinase activity,and further activates the AKT/GSK-3β/β-catenin axis.