The Mechanism Of NAT10 Promoting Helicobacter Pylori-Mediated Gastric Carcinogenesis By Regulating GNA13 MRNA Acetylation

BackgroundGastric cancer is one of the most common malignant tumors of gastrointestinal tract,and Helicobacter pylori(Hp)infection is an important pathogenic factor of gastric cancer.mRNA stability and translation efficiency can be regulated by N4acetylcytidine(ac4C)modification.Recent studies have shown that abnormal mRNA ac4C modification is related to tumor development.However,there are few reports on whether Helicobacter pylori mediated the occurrence of gastric cancer by regulating mRNA ac4C modification.Therefore,it is of great significance to study the effect of mRNA ac4C modification on the occurrence and development of gastric cancer mediated by Helicobacter pylori.AimsThis study aims to explore the role and mechanism of abnormal mRNA ac4C modification in the occurrence and development of gastric cancer mediated by Helicobacter pylori,search for new targets for the diagnosis and treatment of gastric cancer,and clarify the specific molecular mechanism of Helicobacter pylori infection upregulating NAT10 expression,mediating ac4C modification of GNA13 mRNA,and promoting the occurrence and development of gastric cancer.MethodsWe evaluated the correlation of NAT-10 expression levels with clinical information by immunohistochemical(IHC)and TCGA database.The expression level of NAT10 in gastric cancer and its adjacent tissues was detected by qRT-PCR.The effects of NAT 10 on proliferation and metastasis of gastric cancer in vitro were investigated by cell biological function experiments.qRT-PCR and western blot were used to detect the effect of Helicobacter pylori on the expression of NAT10 in gastric cancer cells.CCK8 and Transwell assay were used to detect the effect of Helicobacter pylori on the proliferation and migration of gastric cancer cells,and the response test after NAT10 knockdown.The effect of NAT10 on proliferation and metastasis of gastric cancer in vivo was investigated by subcutaneous tumor formation and caudal vein metastasis in nude mice.AcRIP-seq,RIP and RNA stability tests were used to elucidate that NAT 10 affects GNA13 mRNA expression by combining with ac4C modification in gastric cancer.The effect of GNA13 on the proliferation and migration of gastric cancer cells was tested by cell function experiments,and the functional rescue experiment was conducted after overexpression of NAT 10 and knocking down GNA13.Results1.NAT10 is highly expressed in gastric cancer tissues and the high expression is associated with poor prognosisThe results of TCGA database,immunohistochemical staining and qRT-PCR showed high expression of NAT 10 in gastric cancer tissues,and high expression of NAT 10 was associated with poor prognosis in patients with gastric cancer.2.Knockdown of NAT10 inhibits cell proliferation and migrationCCK8,EdU,clonogenic assays and Transwell experiments showed that NAT10 knockdown inhibited the proliferation and migration of gastric cancer cells,and overexpression of NAT 10 promoted the proliferation and migration of gastric cancer cells.3.Knockdown of NAT10 inhibits the growth and metastasis of gastric cancer in vivoThe results of subcutaneous tumor formation and caudal vein injection in nude mice showed that stable NAT 10 knockdown inhibited the growth and metastasis of gastric cancer.4.NAT10 expression is upregulated by Helicobacter pylori infection of gastric cancer cellsTwo standard strains of Helicobacter pylori Hp26695 and Hp11637 were used to infect gastric cancer cells MKN-45 and MGC-803 with concentration and time gradient.Results showed that the expression of NAT 10 was increased in a concentrationdependent and time-dependent manner.5.Helicobacter pylori promotes the proliferation and metastasis of gastric cancer cells by regulating NAT10Helicobacter pylori infection promoted the proliferation and migration of gastric cancer cells,and knocking down NAT 10 partially inhibited the proliferation and migration of gastric cancer cells caused by Helicobacter pylori infection.6.NAT10 regulates GNA13 mRNA ac4C modificationUsing acRIP-seq to analyze the changes of mRNA ac4C modification by NAT 10 knockdown in MKN-45 gastric cancer cells,the candidate molecule GNA13 was screened out.It was determined by acRIP-qPCR and RIP-qRCP that NAT 10 regulated the ac4C modification of GNA13 mRNA,and the expression level of GNA13 was down-regulated after NAT-10 knockdown,indicating that NAT10 regulated the expression of GNA13 by regulating the ac4C modification level of GNA13 mRNA.7.Knockdown of GNA13 inhibits the proliferation and migration of gastric cancer cells,and NAT10 plays a role in promoting the proliferation and migration of gastric cancer cells by regulating GNA13EdU,clonogenic assays and Transwell experiments showed that knockdown GNA13 inhibited the proliferation and migration of gastric cancer cells.Clonogenic assays and Transwell experiments showed that knockdown GNA13 partially rescued the enhancement of proliferation and migration of gastric cancer cells caused by overexpression of NAT10.8.NAT10 promotes the proliferation and migration of gastric cancer cells by regulating GNA13 and activating the GNA13\RhoA\β-catenin pathwayThrough the analysis of String and KEGG pathway,it was found that GNA13 could bind Rho-GEF to regulate RhoA/β-catenin and play a role in promoting cancer.When GNA13 is knocked down,RhoA expression is down-regulated and β-catenin expression is down-regulated.The expression of GNA13,RhoA and β-catenin was also down-regulated by NAT10knockdown.ConclusionHelicobacter pylori infection of gastric cancer cells up-regulates the expression of NAT10.NAT10 is highly expressed in gastric cancer tissues and is associated with poor prognosis in patients with gastric cancer.NAT10 promotes the proliferation and migration of gastric cancer cells in vivo and in vitro.NAT10 regulates the expression of GNA13 by regulatingac4C modification of GNA13 mRNA.NAT10 promotes the proliferation and migration of gastric cancer cells by regulating GNA13,and activates the GNA13\RhoA\β-catenin pathway to promote the progression of gastric cancer.Therefore,our study provides potential strategies for the treatment of gastric cancer.