The Mechanism Of Helicobacter Pylori-Mediated M6A Modification Of Lnc-PLCB1 In The Occurrence And Development Of Gastric Cancer

BackgroundGastric cancer is one of the most common malignant tumors in the world,and its occurrence and progression are affected by Helicobacter pylori infection.Studies have shown that lncRNA can participate in the occurrence and development of gastric cancer,and its expression is affected by Helicobacter pylori infection and m6A modification.M6A modification of RNA molecules can be involved in the occurrence and development of gastric cancer.Previous research results of our group showed that Helicobacter pylori could regulate the expression of m6A modification enzyme METTL14,suggesting that m6A modification of lncRNA may be involved in the occurrence of gastric cancer mediated by Helicobacter pylori.Therefore,this paper explores whether Helicobacter pylori can regulate the expression of lncRNA by affecting its m6A modification,and then mediates the occurrence and development of gastric cancer.AimsThe purpose of this study is to explore the key role and related mechanism of lncRNA m6A modification in the occurrence and development of gastric cancer mediated by Helicobacter pylori,so as to provide new strategies and theoretical basis for the treatment of gastric cancer.MethodsGastric cancer cells were infected with Hp26695 for MeRIP-seq and RNA-seq,and lnc-PLCB1 was selected by qRT-PCR and MeRIP-qPCR.The subcellular localization of lnc-PLCB1 was determined by nucleocytoplasmic separation and FISH assay.The regulatory mechanism of METTL14 on lnc-PLCB1 was determined by qRTPCR,dual-luciferase reporter assay and actinomycin D assay.A series of functional experiments were conducted to detect the effects of lnc-PLCB1 on the proliferation and migration of gastric cancer cells in vitro.The effects of lnc-PLCB1 on the growth and metastasis of gastric cancer cells in vivo were in vestigated by tumor xenograft models and metastasis models of nude mice.Pull down,RIP and FISH/IF co-staining experiments proved that lnc-PLCB1 specifically binds to DDX21 protein.The influence of lnc-PLCB1 on DDX21 protein level was detected by western blot assay.Western blot and qRT-PCR experiments were used to explore the influence of DDX21 on its downstream molecules CCND1 and Slug,and relevant rescue experiments were conducted.Results1.Helicobacter pylori affects the expression of lnc-PLCBl through METTL14 The results of RNA methylation sequencing(MeRIP-seq)and RNA-seq showed thacter pylori infection could regulate the m6A modification and expression level of lncRNA in gastric cancer cells.Further analysis and screening of sequencing results showed that Helicobacter pylori could regulate the expression of lnc-PLCB1 through METTL14.2.METTL14 regulates the expression of lnc-PLCBl in an m6A-dependent mannerMethylated RNA immunoprecipitation coupled qRT-PCR(MeRIP-qPCR)results showed that overexpression of METTL14 could significantly increase the m6A modification of lnc-PLCB1 in GC cells.The results of dual-luciferase reporter assay and overexpression of METTL14-WT/Mut plasmids showed that METTL14 affected the expression of lnc-PLCB1 in an m6A-dependent manner.3.Lnc-PLCBl inhibits the proliferation and migration of gastric cancer cells in vitroInterference of lnc-PLCB1 expression promoted the proliferation and migration of gastric cancer cells,while overexpression of lnc-PLCB1 shows the opposite effect.Consistently,lnc-PLCB1 overexpression rescued the effect of METTL 14 knockdown on cell proliferation and migration.4.Down-regulation of lnc-PLCB1 promotes the growth and metastasis of gastric cancer cells in vivo.Lnc-PLCB1 knockdown promoted the growth and metastasis of gastric cancer cells in nude mice.5.Transcription factor IRF2 promotes the expression of lnc-PLCBl and is regulated by Helicobacter pylori infectionTranscription factor IRF2 could specifically enhance the promoter activity of lncPLCB1 and regulate the expression level of lnc-PLCB1,while its own expression was also affected by Helicobacter pylori infection.Rescue experiments showed that Helicobacter pylori infection could reduce the expression of lnc-PLCB1,while overexpression of IRF2 could rescue the down-regulation effect of Helicobacter pylori infection on lnc-PLCB 1.6.Lnc-PLCBl binds to DDX21 protein and exerts its anti-cancer effect through DDX21Lnc-PLCB1 could bind to DDX21,which is the downstream target molecule of lnc-PLCB1.Knockdown of DDX21 inhibited the proliferation and migration of gastric cancer cells in vitro,and rescue experiments showed that lnc-PLCB1 inhibited the proliferation and migration of gastric cancer cells through DDX21.7.Lnc-PLCB1 down-regulates the expression of CCND1 and Slug through DDX21Western blot and qRT-PCR showed that lnc-PLCB1 could down-regulate the protein level of DDX21 but did not affect its RNA level.DDX21 could promote the expression of CCND1 and Slug.Meanwhile,rescue experiment showed that lncPLCB1 down-regulates the expression of CCND1 and Slug by DDX21.ConclusionHelicobacter pylori infection down-regulates the expression of lnc-PLCB1 through transcriptional regulation by IRF2 and m6A modification mediated by METTL14.Then lnc-PLCB1 promotes the protein expression level of DDX21,and subsequentially up-regulates CCND1 and Slug expression,thus promoting the occurrence and development of gastric cancer.