Preliminary Study On The Role Of Down-regulation Of ASH2L Expression In NF-?B Signaling Pathway Induced By Helicobacter Pylori Infection

Objectives The siRNA technology-ASH2 L expression vector was used to transiently transfect AGS gastric adenocarcinoma cells to achieve down-regulation of ASH2 L expression in AGS cells.siRNA technology was used to down-regulate the expression of ASH2 L in AGS cells infected with Helicobacter pylori(Hp),and the effect of down-regulation of ASH2 L expression on the activation of NF-?B signaling pathway in Hp-infected AGS cells was revealed,revealing that ASH2 L is associated with Hp infection.The role of disease provides a theoretical basis for the diagnosis and prevention of Hp infection-related diseases.MethodssiRNA technology was used to down-regulate the expression of ASH2 L in AGS cells,and AGS cells were infected with Hp.The expression of ASH2 L and NF-?B signaling pathway-related protein molecules was detected by immunoblotting.NF-?B target gene expression was detected by enzyme-linked immunosorbent assay.In this study,the effect of down-regulation of ASH2 L expression on activation of NF-?B signaling pathway in Hp-infected AGS cells was studied.1.Design and selection of ASH2L-siRNA sequence(1)Design of ASH2L-siRNA sequence: According to the design principle ofsiRNA,design 3 pairs of siRNA sequences and 1 pair of siRNA NC sequencesfor ASH2 L target sequences,named ASH2L-homo-251,ASH2L-homo-1455,ASH2L-homo-1564 and siRNA-NC.(2)Real-time fluorescence quantitative PCR was used to detect the silencingeffect of ASH2L-siRNA sequence: AGS cells were cultured to prepare cellsuspension.The above three pairs of siRNA and siRNA-NC were wrappedwith lipofectamine 2000,transfected into AGS cells,and siRNA silencingASH2L experimental group and siRNA-NC negative control group wereestablished.After 15 min of transfection,the cells of each group were collectedand RNA was extracted and the silencing effect of siRNA on ASH2 L wasdetected by real-time PCR.2 Effect of ASH2 L expression down-regulation on activation of NF-?B signaling pathway in Helicobacter pylori Hp-infected AGS cells(1)Down-regulation of ASH2 L expression in AGS cells: The expression ofASH2L in AGS cells was down-regulated by the above siRNA technology,and the siRNA-ASH2 L experimental group and the siRNA-NC control groupwere established,and the treatment factor(Hp live suspension or cell culturesolution)was added.The culture supernatants and cells of each group werecollected at 15 min,45 min,75 min,and 105 min,and the silencing effect ofASH2L in AGS cells was detected by real-time PCR and Western blot.(2)Western blotting was used to detect the expression of NF-?B signalingpathway-related protein molecules: the collected cells were sonicated andsubjected to SDS-PAGE electrophoresis.After transfection,specific hybridantibodies were used to detect the immune hybridization signals,and p65,I?B?,H3 and H3K4me3 were detected.The expression of different treatmentfactors at different time points of the group of cells.Western blotting wasused to detect the expression of NF-?B signaling pathway-related proteinmolecules: the collected cells were sonicated and subjected to SDS-PAGEelectrophoresis.After transfection,specific hybrid antibodies were used todetect the immune hybridization signals,and p65,I?B?,H3 and H3K4me3were detected.The expression of different treatment factors at different timepoints of the group of cells.(3)Enzyme-linked immunosorbent assay(ELISA)was used to detect theexpression of NF-?B target gene: the expression of IL-8 and TNF-? in thesupernatant culture supernatant collected by the above method was detected,and the different treatment factors were detected.The expression of I?B? ateach time point in each group of cells.Results1.Selection and optimization of ASH2L-siRNA sequence(1)Optimization of transfection conditions: AGS cells were confluent to 70%for transfection.The ratio of siRNA-ASH2L/lipofectamine 2000 tosiRNA-NC/lipofectamine 2000 was 3:2,and the transfection efficiency washighest after transfection for 48 hours.(2)Quantitative PCR method was used to detect the effect of siRNA silencingASH2L: ASH2L-homo-251,ASH2L-homo-1455,ASH2L-homo-1564 were usedas experimental group,siRNA-NC was used as control group,andASH2L-homo-251 was down-regulated.The expression of ASH2 L was betterthan that of ASH2L-homo-1455 and ASH2L-homo-1564.Therefore,ASH2L-homo-251 was used as the siRNA-ASH2 L experimental group,andsiRNA-NC was used as the control group for subsequent experiments.2.Results of down-regulation of ASH2 L expression on activation of NF-?B signaling pathway in Hp-infected AGS cells(1)Detection of siRNA-ASH2 L in silencing ASH2 L in infected and uninfectedHp AGS cellsThe results of real-time PCR showed that ASH2L-homo-251 was used as thesiRNA-ASH2 L experimental group and siRNA-NC was used as the controlgroup to study the down-regulation of AHS2 L under the conditions of Hpinfection and Hp uninfected.In the siRNA-ASH2 L group without Hp infection,the expression level of ASH2 L of AGS cells at each time point was lower thanthat of siRNA-NC control group at each time point,the difference wasstatistically significant;siRNA-ASH2 L in Hp infection The expression level ofASH2L of AGS cells in the experimental group was lower than that of thecorresponding time point siRNA-NC control group,and the difference wasstatistically significant.It is indicated that the expression of ASH2 L in AGS cellscan be down-regulated by siRNA technology under Hp-infected and uninfectedconditions.Western blot analysis showed that ASH2 L was expressed in siRNA-ASH2Lexperimental group and siRNA-NC control group with different treatmentfactors: in AGS cells not infected with Hp,the expression level of ASH2 L insiRNA-ASH2 L experimental group was significantly lower than that at eachtime point.In the siRNA-NC control group,the difference was statisticallysignificant.In the cells infected with Hp,the expression level of ASH2 L in theexperimental group was significantly lower than that in the siRNA-NC controlgroup,and the difference was statistically significant.It was shown that theexpression of ASH2 L was down-regulated in both AGS cells infected anduninfected with Hp.At the same time,the expression of ASH2 L in the controlgroup infected with Hp was significantly higher than that in the uninfected Hpcontrol group and the uninfected Hp siRNA-ASH2 L experimental group.Thedifference was statistically significant.(2)Western blot analysis of expression of NF-?B signaling pathway-relatedprotein moleculesThe expression of p65: p65 in the uninfected HP siRNA-ASH2Lexperimental group at each time point than the siRNA-NC control group,thedifference was statistically significant.In the cells infected with Hp,theexpression levels of p65 in the siRNA-ASH2 L experimental group weresignificantly lower than those in the siRNA-NC control cells at the correspondingtime points,and the difference was statistically significant.The expression of p65in the cells infected with Hp was significantly higher than that of the control cellsinfected with Hp and the siRNA-ASH2 L group without Hp infection.Thedifference was statistically significant.The expression of I?B?: I?B? was lower than that of siRNA-NC controlgroup in the siRNA-ASH2 L experimental group without Hp infection,and thedifference was statistically significant.In Hp-infected cells,the expression ofI?B? in the siRNA-ASH2 L experimental group was significantly lower than thatin the siRNA-NC control cells at the corresponding time points,and thedifference was statistically significant.The expression of I?B? in the cellsinfected with Hp was significantly higher than that of the control cells infectedwith Hp and the siRNA-ASH2 L group without Hp infection.The difference wasstatistically significant.The expression of H3 was lower than that of siRNA-NC control group in thesiRNA-ASH2 L experimental group without Hp infection,and the difference wasstatistically significant.In the cells infected with Hp,the expression levels of H3in the siRNA-ASH2 L experimental group were significantly lower than those inthe siRNA-NC control cells at the corresponding time points,and the differencewas statistically significant.The expression level of H3 in the cells infected withHp was significantly higher than that of the control cells without Hp and thesiRNA-ASH2 L group without Hp infection.The difference was statisticallysignificant.The expression of H3K4me3: H3K4me3 expression in the siRNA-ASH2Lexperimental group without Hp was lower than that in the siRNA-NC controlgroup,the difference was statistically significant.In the cells infected with Hp,the expression levels of H3K4me3 in the siRNA-ASH2 L experimental groupwere significantly lower than those in the siRNA-NC control cells at thecorresponding time points,and the difference was statistically significant.Theexpression of H3K4me3 in the cells infected with Hp was significantly higherthan that of the control cells infected with Hp and the siRNA-ASH2 L groupwithout Hp infection.The difference was statistically significant.(3)Results of NF-kB target gene expression detected by ELISAThe results of IL-8 assay showed that the expression of IL-8 in thesiRNA-ASH2 L group without Hp was slightly lower than that of the siRNA-NCcontrol cells that were not infected with Hp at the corresponding time points.Thedifference was not statistically significant.significance.In the Hp-infected cells,the expression levels of IL-8 in the siRNA-ASH2 L experimental group werelower than those in the siRNA-NC control cells at the corresponding time points,and the difference was statistically significant.The expression level of IL-8 in thecells infected with Hp was significantly higher than that of the control cells notinfected with Hp and the siRNA-ASH2 L experimental group not infected withHp at each time point.The results of TNF-? assay showed that the expression of TNF-? in thesiRNA-ASH2 L group without Hp was slightly lower than that of the siRNA-NCcontrol cells that were not infected with Hp at the corresponding time points.Thedifference was not statistically significant.significance.In Hp-infected cells,theexpression levels of TNF-? in the siRNA-ASH2 L experimental group were lowerthan those in the siRNA-NC control cells at the corresponding time points,andthe difference was statistically significant.The expression levels of TNF-? in thecells infected with Hp control at each time point were significantly higher thanthose of the control cells not infected with Hp and the siRNA-ASH2Lexperimental group not infected with Hp.The results of I?B? assay showed that the expression of I?B? in thesiRNA-ASH2 L group without Hp was significantly lower than that of thesiRNA-NC control cells that were not infected with Hp at the corresponding timepoints.The difference was statistically significant.In Hp-infected cells,theexpression levels of I?B? in the siRNA-ASH2 L experimental group were lowerthan those in the siRNA-NC control cells at the corresponding time points,andthe difference was statistically significant.The expression level of I?B? in thecells infected with Hp control at each time point was significantly higher thanthat of the control cells not infected with Hp and the siRNA-ASH2 L experimentalgroup not infected with Hp.Conclusion 1.The designed ASH2L-siRNA sequence was transfected with lipofectamine 2000to down-regulate ASH2 L expression in AGS cells.2.In the control group,the expression of ASH2 L increased with the prolongation ofHp-infected AGS cells,indicating that Hp infection can up-regulate ASH2Lexpression and activate NF-?B signaling pathway.3.The expression levels of NF-?B signaling pathway-related protein molecules(P65,I?B?,H3,H3K4me3)in Hp control cells were significantly higher than those ofcontrol cells not infected with Hp and those without Hp infection.,indicating thatHp infection can lead to the activation of NF-?B signaling pathway in AGS cells;and the expression levels of these NF-?B signaling pathway-related proteinmolecules in Hp-infected siRNA-ASH2 L experimental group are significantlylower than the corresponding time points.Hp-infected siRNA-NC control cellsindicated that down-regulation of ASH2 L expression reduced the activation ofNF-?B signaling pathway after Hp infection.4.The expression of NF-?B target genes(IL-8,TNF-?,I?B?)in the control cellsinfected with Hp increased with the prolongation of infection time,indicating thatHp infection can increase the expression of NF-kB target gene;The expressionlevels of these target genes in Hp-infected siRNA-ASH2 L experimental groupwere significantly lower than those in Hp-infected siRNA-NC control cells at thecorresponding time point,indicating that down-regulation of ASH2 L expressioncan make NF-H-infected NF-The expression of ?B-related target genes IL8,TNF-?,and I?B? was down-regulated.5.This study preliminarily showed that down-regulation of ASH2 L expressionreduced the activation of NF-?B signaling pathway after Hp infection,anddown-regulated the expression of NF-?B-related target genes IL-8,TNF-? andI?B?.