The Role And Mechanism Of Helicobacter Pylori In Regulating The M~6A Modification Of CircZRANB1 To Promote The Progression

Background:Gastric cancer(GC)is a highly malignant disease with poor prognosis and low 5-year survival rate.Helicobacter pylori(Hp)infection is considered to be an important risk factor for malignant transformation of gastric mucosal cells.However,the mechanism by which Hp promotes the malignant transformation of gastric mucosal cells is not fully understood.Therefore,it is very important to explore the molecular mechanism of Hp mediated malignant transformation of gastric mucosal cells and find the key target molecules in the progression of gastric cancer to increase the survival rate of patients with gastric cancer.Circular RNAs(circRNAs)and m6A methylation modification of RNA are the current frontier hot topics.The dysregulated expression of circRNAs or abnormal m6A modification of RNA are closely related to tumorigenesis.Whether Hp regulates m6A modification of circRNAs in host cells has not been reported.In this study,we explored the new pathogenic mechanism of Hp from the perspective of Hp regulating the expression of circZRANB1 and its m6A methylation modification,and explored the biological function and regulatory mechanism of circZRANB1 in gastric cancer cells,and evaluated its potential value as an early diagnosis and therapy of gastric cancer.Methods:1.The effect of Hp infection on the protein expression of m6A modification related enzymes in gastric cancer cells was detected by Western Blot.2.qRT-PCR and MeRIP-qPCR were used to detect the expression level of circZRANB 1 and the m6A methylation modification of circzRANB1 RNA.3.qRT-PCR,MeRIP-qPCR,RIP,Nuclear and cytoplasmic separation experiments and immunofluorescence assay were used to investigate the regulation of METTL3 on the expression and m6A modification of circZRANB1 and the mechanism of METTL3-mediated m6A modification of circZRANB1 in regulating the expression level of circular RNA.qRTPCR,RNase R and ActD treatment were used to detect the stability of circZRANB 1.4.EdU,CCK8,colony formation,and Transwell assays were used to detect the effects of circZRANB1 overexpression or interfering with the proliferation and migration of gastric cancer cells.5.RNA pull-down,qRT-PCR,RIP and other experiments were used to explore the interaction between circZRANB1 and m6A "reading protein" IGF2BP2,the regulation and mechanism of the complex formed by circzRANB1 and m6A on the downstream target molecules.EdU and Transwell experiments were used to verify the biological function recovery experiment to verify the mechanism of circZRANB1.Results:1.Regulation of Hp on the expression of m6A modification-related enzymes in gastric cancer cells:compared with adjacent tissues,METTL3 transcript was significantly increased in gastric cancer tissues and negatively correlated with the survival time of gastric cancer patients.Hp infection can raise the protein level of METTL3 in gastric cancer cells.2.Effect and mechanism of Hp on the expression level of circZRANB1 and m6A modification:Hp infection can raise the expression level of circZRANB1 and m6A modification in gastric cancer cells.The circZRANB1 modified by m6A could be recognized and bound by the m6A reading protein YTHDC1,promoting the nuclear export of circZRANB 1.3.CircZRANB1 is more stable than linear molecule:after RNA and gastric cancer cells were treated with RNase R and ActD for different periods of time,the expression level of linear molecule ZRANB1 significantly decreased,while the expression level of circZRANB1 changed little,indicating that circZRANB1 was more stable than linear ZRANB1.4.CirCZRANBl biological function:overexpression of circZRANB1 significantly promoted the proliferation and metastasis of gastric cancer cells,while knockdown of circZRANB 1 had opposite biological functions.5.Mechanisms by which CircZRANB1 functions:IGF2BP2 could specifically bind to circZRANB1,and METTL3 could promote the binding of circZRANB1 and IGF2BP2,and the complex formed by them could significantly increase the mRNA and protein levels of SERPINE2.6.CircZRANB1 functions through its downstream molecules SERPINE2:cooverexpression of SERPINE2 in the knockdown cells reversed the biological functions mediated by circZRANB 1 knockdown.Conclusion:Helicobacter pylori infection can increase the m6A modification and expression level of circZRANB 1.The m6A modification of circZRANB1 can be recognized and bound by YTHDC1 in the nucleus and promote the transport of circzranB1 to the cytoplasm.CircZRANB1 modified by m6A in the cytoplasm can increase the interaction with IGF2BP2,and the complex formed by circzRANB 1 and IGF2BP2 can increase the stability and expression of SERPINE2,thereby promotion of gastric cancer cell proliferation and migration.This study reveals the pathogenic mechanism of Hp from the perspective of regulating the m6A methylation of circular RNA,and provides a theoretical basis for the development of new antigastric cancer drugs targeting the circular RNA circZRANB1.