Helicobacter Pylori-mediated FTO Decreases Rnf4expression Via M~6A Modification And Participates In DNA Damage Response,Migration And Invasive In Infected Cells

Objective:The purpose of this study was to investigate the role of Helicobacter pylori(Hp)infection in m~6A methylation modification in gastric epithelial cells,as well as the effects of Hp-mediated FTO regulating RNF4 expression on DNA damage response,invasive and migration of infected cells.Methods:1.The effect of Hp infection on cellular m~6A methylation modification and its mediating effect of FTO on the invasion and migration of infected cells:(1)Hp infected gastric epithelial cells GES-1 and AGS at MOI of 60:1 for 24h,then total RNA and total protein was extracted,and m~6A RNA dot blot assay was performed to detect the level of total cellular RNA m~6A modification,and real-time fluorescence quantitative PCR and Western blot were used to detect the expression of m~6A methylation modification related enzymes such as FTO,METTL3 and YTHDF2.(2)Bioinformatic analysis was used to predict expressions and prognosis of FTO,METTL3 and YTHDF2,and the relationship between FTO expression and clinicopathological parameters of gastric cancer and functional enrichment in gastric cancer.(3)Using immunohistochemical techniques to detect FTO expression in human gastric cancer tissues,and in Hp-infected Mongolian gerbil gastric tissues.(4)A stable transgenic cell line with knockdown FTO gene was constructed and FTO expression was detected by Western blot;the effect of cellular m~6A methylation modification was detected by m~6A dot blot assay after Hp infection of FTO knockdown cells.(5)Cell scratch assay and Transwell assay were used to detect the effect of migration ability and invasion ability of Hp-infected FTO knockdown cells.2.Effect of Hp-mediated FTO on RNF4 expression via m~6A demethylation modification:(1)Bioinformatic analysis was used to predict target genes of FTO m~6A modification.(2)While Hp had infected FTO knockdown cells,Western blot was used to detect RNF4 expression levels and Me RIP-q PCR was performed to detect RNF4 m~6A levels.3.The role of RNF4 in Hp-induced DNA damage response and invasive migration in gastric epithelial cells:(1)Immunohistochemical and bioinformatics analysis were performed to detect the expression of RNF4 and to predict RNF4functional enrichment in gastric cancer.(2)RNA interference technique down-regulated RNF4 gene expression in GES-1 and AGS cells,then using Western blot to verify the expression level of RNF4,and comet assay was used to detect cellular DNA damage,Western blot was performed to detect the expression of DNA damage key proteins such asγ-H2A.X,P53 and RAD51.(3)A stable transgenic cell line overexpressing RNF4 was constructed and infected with Hp.The expressions of RNF4 and key DNA damage repair proteins P53 and RAD51 were detected by Western blot,and the expression and localization ofγ-H2A.X and RAD51 were detected by immunofluorescence,and the cellular DNA damage was detected by comet assay.(4)Using cell scratch assay and Transwell assay to detect knockdown RNF4 and Hp infection overexpressing RNF4 in gastric epithelial invasion migration ability.Results:1.Hp upregulation of FTO gene expression decreased total m RNA m~6A methylation levels in infected cells and promoted migration and invasion of infected cells.(1)Compared with the uninfected group,Hp-infected gastric epithelial cells showed decreased levels of total m RNA m~6A methylation modification,and increased FTO m RNA and protein expression,and decreased METTL3 and YTHDF2 m RNA and protein expression.(2)Bioinformatic analysis showed that FTO and YTHDF2m RNA expression was higher in gastric cancer tissues than in normal tissues,and METTL3 m RNA expression was lower than in normal tissues;high FTO expression was negatively correlated with the prognosis of gastric cancer patients,and positively correlated with pathological parameters such as T-stage,M-stage and AJCC stage of gastric cancer patients;FTO was involved in regulating m RNA methylation,RNA stability,DNA damage repair,cellular value addition,metabolism and other processes.(3)Immunohistochemical results showed that the expression level of FTO protein in gastric cancer tissues was significantly higher than that in para-cancerous tissues,and the expression of FTO in gastric tissues of Hp-infected Mongolian gerbils was also higher than that in control tissues.(4)The knockdown FTO cell lines were successfully constructed and the total RNA m~6A levels were significantly higher in gastric epithelial cells with knockdown FTO compared to the null group;compared to knockdown FTO cells,Hp-infected FTO knockdown cells showed higher FTO expression and lower total cellular m RNA m~6A levels.(5)Hp infection significantly increased the migratory invasive ability of cells compared with the non-infected group;knockdown FTO gene inhibited the migratory and invasive ability of cells compared with the null group,and knockdown FTO inhibited the migratory invasive ability of Hp-infected cells.2.Hp-mediated FTO down-regulates RNF4 expression through m~6A methylation modification.(1)The RM2 Target website predicted the presence of m~6A modification sites in RNF4,and an m~6A modification target protein of FTO.(2)Knockdown of FTO caused an increase in RNF4 m RNA m~6A levels and upregulated RNF4 protein expression compared to the null group.(3)Compared to knockdown FTO cells,Hp infection of FTO knockdown cells with elevated FTO expression caused downregulation of RNF4 m~6A levels and decreased RNF4 expression levels,which suggested that Hp could mediate FTO downregulation of RNF4 expression through de-m~6A methylation modifications.3.Hp down-regulation of RNF4 expression was involved in DNA damage response and invasive migration of gastric epithelial cells.(1)Bioinformatic analysis showed that RNF4 was lowly expressed in gastric cancer tissues;RNF4 was mainly involved in cellular stress response and DNA damage repair,and thus in tumorigenesis and progression.(2)Reduced RNF4 expression in sh RNF4-transfected GES-1 and AGS cells;longer DNA trailing in Hp-infected cells compared to non-infected group;aggravated genomic DNA damage in Hp-infected RNF4 knockdown cells compared to Hp-infected null group;reduced expression of P53 and RAD51,key proteins for DNA damage repair,in cells with knockdown RNF4 compared to null group.(3)RNF4 expression was elevated in GES-1 and AGS cells overexpressing the RNF4 gene;compared with the non-infected group,DNA trailing was longer and RAD51 and P53 stability was reduced in Hp-infected cells;compared with the null group,overexpression of RNF4 upregulated RAD51 and P53 protein levels;compared with the RNF4 overexpression group,Hp-infected cells overexpressing the RNF4 gene compared with the RNF4 group,Hp-infected cells overexpressing RNF4gene showed reduced DNA damage,accelerated aggregation ofγ-H2AX and RAD51in the nucleus,and stable expression of P53 and RAD51,suggesting that overexpression of RNF4 gene could reduce DNA damage in Hp-infected cells.(4)Compared with the null group,knockdown of RNF4 promoted migration and invasion of gastric epithelial cells,and overexpression of RNF4 reduced cell invasion and migration ability;compared with the Hp-infected null group,Hp-infected overexpression of RNF4 cells had reduced invasion and migration ability.Conclusions:(1)Hp may reduce m~6A levels in infected cells through upregulation of FTO expression and promote cell migration and invasion.(2)Hp could down-regulate RNF4 expression through FTO de-m~6A modification.(3)Hp down-regulates RNF4 expression in response to DNA damage and invasive and migration in gastric epithelial cells.