Study On The Effect Of Silencing ASH2L And IQGAP1 On The Biological Effects Of AGS Gastric Cancer Cells And Its Role In HP

Objective:Early experimental studies in our laboratory showed that under the conditions of Helicobacter pylori(Hp)infection,the expression of ASH2 L in AGS cells increased,and there was an interaction between ASH2 L and IQGAP1.This study intends to silence ASH2 L and IQGAP1 genes to explore their effects on the proliferation,migration,invasion and apoptosis of human gastric cancer AGS cells and analyze their mechanisms of action in Hp infection-related diseases.Methods:1.The effect of silencing ASH2 L and IQGAP1 genes on the biological effects of gastric adenocarcinoma AGS cells: Silencing the expressions of ASH2 L gene and IQGAP1 gene in gastric adenocarcinoma AGS cells with si RNA technology,using real-time fluorescent quantitative PCR and Western blot to detect the silencing effect;using cck-8 to detect the proliferation of gastric cancer cells;Scratch healing test and transwell test to detect changes in gastric cancer cell migration and invasion ability;TUNEL experiment and flow cytometry to detect the apoptosis of gastric cancer cells.2.Transcriptome sequencing(RNA-sequencing,RNA-seq)technology to study the role and mechanism of silencing ASH2 L and IQGAP1 genes and Hp infection in the occurrence and development of gastric cancer: After down-regulating the expression of ASH2 L gene and IQGAP1 gene in gastric adenocarcinoma AGS and normal gastric mucosal epithelial cells GES-1,add Hp infection factor treatment to prepare three biological replicate RNA samples of Hp-infected and Hp-uninfected AGS and GES-1 cells respectively.Use RNA-seq technology to perform high-throughput sequencing of the transcriptome,perform bioinformatics analysis on sequencing data,perform corresponding enrichment analysis(GO,KEGG)for differential genes,and then apply online software for protein interaction(Protein-Protein Interaction,PPI)network analysis,In order to explore the mechanism of the influence of ASH2 L and IQGAP1 on the biological effects of cells,analyze the possible mechanisms of Hp infection induction and gastric cancer and the role and mechanism of ASH2 L gene and IQGAP1 gene in the NF-κB signal pathway activated by Hp infection.Results:1.Results of the effects of silencing ASH2 L and IQGAP1 genes on the biological effects of AGS cells(1)Compared with the control group,the m RNA level and protein expression of the ASH2 L gene in the ASH2 L silence group and the IQGAP1 gene in the IQGAP1 silence group were significantly down-regulated(P values were less than 0.05).(2)After silencing ASH2 L and IQGAP1 genes,respectively,AGS cells showed diminished proliferative capacity,diminished migratory capacity,diminished invasive capacity,and increased apoptotic rate compared with the control group(all P values were less than 0.05).2.Transcriptome sequencing data analysis results(1)The silencing efficiency of ASH2 L and IQGAP1 genes was higher than 75%in all groups of cells,and all 30 RNA samples submitted for sequencing met the sequencing criteria.All groups were enriched in related differential genes,and the expression difference multiples of differential genes were |log2Fold Change|>1.(2)Analysis of the mechanism of action of silencing ASH2 L gene and silencing IQGAP1 gene on the biological effect of gastric adenocarcinoma AGS cells: The transcriptome sequencing data of silencing ASH2 L gene and silencing IQGAP1 gene in AGS cells were analyzed,and using Wayne analysis,829 differentially expressed genes were found to be jointly affected by silencing the two genes,among which 829 differentially expressed genes were jointly upregulated 160 and co-down-regulated656.Further analysis using GO enrichment analysis revealed that the ANXA3,HBEGF,ITGA3,ETS1,SCARB1,VEGFA,KLF4,ADGRA2,BCAS3,TDGF1,PTPRM,ACVRL1,ANGPT4,HRG were involved in epithelial cell migration;the CHAC1,CASP4,ATF4,XBP1,ERN1,TRIB3,and PMAIP1 were involved in endoplasmic reticulum stress..The KEGG enrichment results showed that the differentially expressed genes caused by silencing two genes are jointly involved in the following biological signaling pathways: m TOR signaling pathway,Apoptosis,Calcium signaling pathway,MAPK signaling pathway,TGF-beta signaling pathway,and VEGF signaling pathway.(3)Analysis of the mechanism of Hp infection on the carcinogenesis of normal gastric epithelial cells GES-1: The differentially expressed gene libraries in Hp-infected and Hp-uninfected GES-1 cells were analyzed,and the differentially expressed gene libraries in AGS cells and GES-1 cells were also analyzed,and further using Wayne analysis,1059 differentially expressed genes were found to be common to both differential gene libraries,of which 160 differentially genes were co-upregulated and 656 were co-downregulated.The common signal pathways related to carcinogenesis enriched by KEGG include: Pathways in cancer,Erb B signaling pathway,p53 signaling pathway,TGF beta signaling pathway,MAPK signaling pathway and Wnt signaling pathway.(4)Role and mechanism of ASH2 L gene and IQGAP1 gene in NF-κ B signaling pathway activated by Hp infection: ASH2 L gene was silenced in AGS cells,and the expression of ASH2 L and ALPK1 gene was significantly down regulated.However,the expression of ASH2 l and ALPK1 gene was not significantly down regulated after Hp infection;The expression of both IQGAP1 and ALPK1 genes were significantly downregulated by silencing IQGAP1 gene in AGS cells,and after adding Hp infection,the expression of IQGAP1 gene was no longer significantly downregulated,while the expression of ALPK1 gene tended to be upregulated,thus it is hypothesized that Hp infection may affect the progression of disease after Hp infection by affecting the expression of ASH2 L gene and IQGAP1 gene to regulate the activity of ALPK1 gene and activate NF-κB signaling pathway.Conclusion:Silencing of ASH2L and IQGAP1 genes was achieved in AGS cells,and the silencing of ASH2 L gene as well as IQGAP1 gene can inhibit the proliferation,migration and invasion of AGS cells,and promoted their apoptosis.ASH2 L and IQGAP1 may regulate cell migration and invasion through the VEGF signaling pathway,and regulate apoptosis through the MAPK signaling pathway;Hp infection may lead to carcinogenesis of normal gastric epithelial cells through pathways in cancer,erb B signaling pathway,p53 signaling pathway,TGF beta signaling pathway,MAPK signaling pathway and Wnt signaling pathway;Hp infection may affect the occurrence and development of Hp infection-related diseases by affecting the expression of ASH2 L and IQGAP1 genes to regulate the activity of ALPK1,which in turn activates the NF-κB signaling pathway.