ASH2L Promotes Gastric Cancer By Regulating The ALPK1/NF-κB Signaling Pathway Activated By Helicobacter Pylori Infection

Objectives:In this study,we wanted to initially define the roles of ASH2L and ALPK1 in the development,progression,diagnosis and prognosis of gastric cancer by bioinformatics analysis of their expression and clinical significance in stomach adenocarcinoma;The dynamic expression of ASH2L and ALPK1 in Hp infected and uninfected gastric cell lines was analyzed by in vitro cell experiments to explore the roles of ASH2L,ALPK1in regulating the NF-κB signaling pathways activated by ALPK1-TIFA induced by Hp infection.Methods:1 Bioinformatics analysis of ASH2L and ALPK1 expression and clinical significance in stomach adenocarcinoma:The TIMER2.0 tool was used to analyze the expression levels of ASH2L and ALPK1 in different tumor tissues;The differential expression of ASH2L and ALPK1 in stomach adenocarcinoma and their correlation with clinicopathological characteristics of patients with stomach adenocarcinoma were analyzed by UALCAN platform;The Kaplan-Meier plotter survival analysis tool was used to evaluate the effects of ASH2L and ALPK1 on the survival of patients with stomach adenocarcinoma;Analysis of the correlation of ASH2L with ALPK1 in stomach adenocarcinoma using the GEPIA tools;Silencing ASH2L using si RNA technology and detecting the effect of silencing ASH2L on ALPK1 expression through quantitative Real-time PCR;The m RNA transcriptome sequencing data of our subject group were subjected to Venn analysis to explore the effect of silencing ASH2L in AGS cells on ALPK1 expression and the effect of Hp infection on ASH2L and ALPK1 expression.2 To analyze the roles of ASH2L and ALPK1 in the NF-κB signaling pathway activated by ALPK1-TIFA in response to Hp infection.(1)Expression of ASH2L and ALPK1 was detected at the m RNA level;Pmss1(Cag A~+)and pmss1(Cag A~-)Hp strains were infected in the AGS and GES lines,respectively,along with TNF-αat concentrations of 1ng/m L and 10ng/m L,respectively Above two kinds of cells were treated to serve as positive control for NF-κB signaling pathway activation.RNA samples were prepared before and after infection and treatment,Using quantitative Real-time PCR technology to detect the relative expression levels of ASH2L and ALPK1 at m RNA level under six different infection time conditions(0min,15min,30min,45min,60min,and 75min),and with an infection time of 60 min NF-κB signaling pathway target genes A20,IκBαAnd TNF-αof the relative expression in cells.(2)Detection of ASH2L and ALPK1 expression at the protein level:Three Hp strains,43504(Cag A~+),pmss1(Cag A~+)and pmss1(Cag A~-),were infected in the AGS and GES with 1ng/m L and 10ng/m L of TNF-α,respectively Both cells were treated to serve as Positive control for NF-κB signaling pathway activation.Protein samples before and after infection and treatment were prepared.When the Hp infection time was 60minutes,cells were treated with 5 different infection complexes(0,10,50,100,and 200)and Western blot technology was used to detect ASH2L,ALPK1,and NF-κB signaling pathway related protein molecules P65,p-P65,IκBαAnd p-IκBαthe level of expression.Similarly,when the infection complex is 100,Western blot technology is used to detect the expression levels of the aforementioned proteins in the cells at the six different infection time points mentioned above.(3)Activation of NF-κB signaling pathways dependent on the ALPK1-TIFA axis and expression of ASH2L and ALPK1 were examined at the protein level:Pmss1(Cag A~+)and pmss1(Cag A~-)Hp strains at a multiplicity of infection of 100 were added to the AGS gastric cell line,concurrent treatment with TNF-αAGS cells were treated to serve as a positive control for NF-κB signaling pathway activation.ASH2L,ALPK1,P65,p-P65,TIFA and p-TIFA protein expression in AGS cells was determined by Western blot analysis 60 min after infection.Results:1 Bioinformatics analysis results(1)Analysis of the differential expression of ASH2L and ALPK1 genes in tumor tissues:In 56 common tumors,ASH2L was differentially expressed in 15 tumors and ALPK1 was differentially expressed in 15 tumors compared with normal tissues adjacent to cancer(P<0.05).(2)Correlation analysis between ASH2L and ALPK1 genes and clinicopathological characteristics of stomach adenocarcinoma patients:compared with normal gastric tissues,ASH2L and ALPK1 genes are highly expressed in stomach adenocarcinoma tissues;ASH2L expression in stomach adenocarcinoma was correlated with patient’s race,age,pathologic grade,tumor stage,lymph node metastasis,and histologic subtype,but not with male or female gender;ALPK1 expression was correlated with patient age,pathological grade,tumour stage,lymph node metastasis and histological subtype,but not with ethnicity or both genders(P<0.05).(3)Analysis of the effect of ASH2L and ALPK1 genes on the survival of patients with stomach adenocarcinoma:The patients with high expression of ASH2L and ALPK1 genes have worse overall survival and post-progression survival than those with low expression(P<0.05).(4)Correlation analysis of ASH2L and ALPK1 gene in patients with stomach adenocarcinoma:there is a positive correlation between ASH2L and ALPK1 gene expression level in stomach adenocarcinoma(P<0.05).(5)Effect of down-regulation of ASH2L expression on ALPK1 expression:the ASH2L gene was successfully silenced in AGS cells,and the expression of ALPK1 was also significantly decreased after down-regulation of ASH2L expression(P<0.05).(6)Venn analysis of m RNA transcriptome sequencing data:the expression of both ASH2L and ALPK1 was significantly downregulated after knockdown of ASH2L in AGS cells,but not after addition of Hp infection factors.2 Roles of ASH2L and ALPK1 genes in the NF-κB signaling pathway activated by ALPK1-TIFA due to Hp infection(1)Expression of ASH2L and ALPK1 were examined at the m RNA level:In both gastric cell lines,the expression of NF-κB signaling pathway target genes A20,IκBαAnd TNF-αwas significantly up-regulated in the Hp infected and TNF-αtreated cells compared with the untreated cells;Compared with the pmss1(Cag A~-)infected group,the pmss1(Cag A~+)infected group resulted in a more significant magnitude of target gene upregulation(P<0.05).Compared with the cells in the no treatment group,after separately infecting the two gastric cell lines with different Hp strains,the ASH2L and ALPK1 expression amounts in the cells all showed a tendency to gradually upregulate with longer infection time.In AGS cells,under the above six different infection time conditions,the expression level of ASH2L in the pmss1(cag A~+)infection group was higher than that in the pmss1(cag A~-)infection group(P<0.05).At the infection time of 60min and 75min,the expression of ALPK1 in the pmss1(Cag A~+)infection group was higher than that in the pmss1(Cag A~-)infection group(P<0.05).Two different TNF-αThe expression amounts of ASH2L and ALPK1 in the two gastric cell lines after concentration treatment did not show obvious changes with time extension.(2)Protein level assay of ASH2L and ALPK1 expression infection of AGS and GES cell lines with three Hp strains,pmss1(Cag A~+),pmss1(Cag A~-),and 43504(Cag A~+),both ASH2L and ALPK1 expression tended to increase with increasing multiplicity of infection or duration of infection in these cells,There is no significant trend in the expression level of P65,IκBαThe expression level of is showing a downward trend,whereas the expression of related protein molecules p-P65 and p-IκBαactivated by NF-κB signaling pathways showed an upregulation;After treatment with two different concentrations of TNF-αASH2L and ALPK1 expression did not change significantly over time in the two gastric cell lines treated,p-p65 and P-IκBαthe expression of circrnas showed an obvious upregulation trend.(3)Activation of the ALPK1-TIFA axis dependent NF-κB signaling pathway and the expression of ASH2L and ALPK1 were examined at the protein level:Compared with the blank group without treatment,the expression of TIFA in the two Hp infected groups with pmss1(Cag A~+)and pmss1(Cag A~-)showed a trend of downregulation,and the expression of p-TIFA and p-P65 showed a trend of upregulation.Conclusion:The ASH2L and ALPK1 genes have potential value as clinical diagnostic,prognostic,and therapeutic targets in stomach adenocarcinoma,Preliminary analysis indicated that the expression amounts of ASH2L and ALPK1 exhibited a positive correlation in gastric adenocarcinoma cells.The expression of ASH2L and ALPK1 in the activation of NF-κB signaling pathways induced by Hp infection both tended to increase with the amount of infected bacteria and increased with the duration of infection,Meanwhile,p-P65,p-IκBαAnd p-TIFA,expression as well as NF-κB signaling pathway target genes A20,IκBαand TNF-αTheir expression was significantly up-regulated.And in TNF-αIn the treated cells,only the ALPK1-TIFA axis dependent NF-κB signaling pathway was activated,and there was no significant change in the expression levels of ASH2L and ALPK1,which suggested that this change in ASH2L and ALPK1 expression was a result of Hp infection.The findings initially suggest that ASH2L associates with ALPK1 in Hp infection induced NF-κB signaling pathway activation,but by what mechanism ASH2L cooperates with ALPK1 to affect the NF-κB The activation of,which in turn plays a role in the development and progression of gastric cancer induced by Hp infection,requires further study.