Screening Of ASH2L Interacting Protein And Differential Expression Related Genes In Helicobacter Pylori Infected AGS Cells

Objective:Co-IP and GST-pulldown were used to identify the proteins interacting with ASH2L.RNA-seq and ChIP-seq were used to analyze the differential expression of AGS cells transcriptome and whole genome before and after Hp infection,and to explore the pathogenesis of ASH2L in Helicobacter pylori(Hp)infection related diseasesMethod:1.Screening and identification of ASH2L interacting protein in Hp infected AGS cells:AGS cells infected with Hp were collected and PBS buffer treated AGS cells were used as blank control to obtain their total proteins.Using Co-IP and GST-pulldown technology,combined with mass spectrometry,Western blot,we screened and identified the interaction proteins with ASH2L in and out of the cells,and bioinformatics analysis of the interaction proteins was carried out.2.Screening and identification of differentially expressed genes in AGS cells under Hp infection and non infection conditions:(1)RNA-seq technique was used to analyze the differentially expressed genes of AGS cell transcriptome before and after Hp infection.Three biological duplicate samples of Hp infected and uninfected AGS cells were collected The total RNA was extracted by Trizol method,the cDNA library was constructed,and next-generation sequencing.Bioinformatics analysis of sequencing data was carried out to annotate differentially expressed genes into biochemical metabolic pathway and signal transduction pathway.(2)The differential expression of genes in AGS cells before and after Hp infection was analyzed by ChIP-seq technique.The Hp infected and uninfected AGS cells were collected and immunocoprecipitated with anti-H3K4me3 antibody.After the construction of DNA library and next-generation sequencing,the sequence was compared to the reference genome,and the Peak related genes were screened by Peak analysis and annotation with MACS2.After a series of bioinformatics analysis,we found the target genes and transcription factors before and after Hp infection,screened out the target genes that were up-regulated and down regulated under the condition of Hp infection and non infection,and annotated these target genes into the biochemical metabolic pathway and signal transduction pathwayResult:1.Screening and identification of ASH2L interacting protein in Hp infected AGS cells:Through the comprehensive analysis of Co-IP and GST-pulldown,WB confirmed that IQGAP1 and hnRNP U can directly interact with ASH2L in Hp infected AGS cells.Bioinformatics analysis showed that IQGAP1 and hnRNP U may participate in NF-kappa B signaling pathway,adherens junction,Regulation of actin cytoskeleto,Proteoglycans in cancer etc.2.Screening and identification of differentially expressed genes in AGS cells under Hp infection and non infection conditions:The results of RNA-seq analysis showed that there were 75 genes differentially expressed between Hp infected AGS cells and non Hp infected AGS cells,which were enriched into 115 pathways,including choline metabolism in cancer,microRNAs in cancer,cancer pathway,transcriptional misregulation in cancer,gastric cancer,gastric acid secretion,epithelial cell signaling in Helicobacter pylori infection,GnRH signaling pathway,NF-kappa B signaling pathway,TNF signaling pathway,Ras signaling pathway,PI3K-Akt signal pathway etc.The results of ChIP-seq analysis showed that there were 76 differentially binding genes between Hp infected AGS cells and non Hp infected AGS cells,and 48 differentially binding up-regulated genes were annotated into three signaling pathways,namely osteoclast differentiation,ribosomeand signaling pathways regulating pluripotency of stem cells;28 differential binding up-regulated genes were annotated into 23 pathways,including:mTOR signaling pathway,cancer pathway,spliceosome,transcriptional misregulation in cancer,Wnt signaling pathway,ribosome and RNA metabolism etcConclusion:1.The expression of ASH2L,hnRNP U and IQGAP1 was up-regulated in Hp infected AGS cells,and ASH2L and hnRNP,respectively There is a direct interaction between u and IQGAP1,among which the interaction between ASH2L and IQGAP1 is the first discovery;at the same time,the three may participate in the activation of NF-kappa B signaling pathway,adherens junction,Regulation of actin cytoskeleto,Proteoglycans in cancer and spliceosome etc.,and play an important role in the development of Hp infection related diseases and gastric cancer.2.Hp infection can change the expression pattern of AGS cell transcriptome and whole genome.After Hp infection,it may affect gastric cancer,gastric acid secretion,epithelial cell signal transduction of Helicobacter pylori infection,GnRH signal pathway,NF-kappa B signaling pathway,Ras signaling pathway,PI3K-Akt signal pathway,TNF signaling pathway,mTOR signal pathway,cancer Pathway,transcriptional misregulation in cancer,Wnt signaling pathway,ribosome and RNA metabolism affect the occurrence and development of Hp infection related diseases.