Identification Of Mutation Sites Of H.pylori Antibiotic-Resistant Genes And Comprehensive Analysis Of CeRNA Network Of Gastric Cancer With H.pylori Infection

Objective: This study focused on two aspects,1.Selecting and identifying mutation sites associated with Helicobacter pylori(H.pylori)resistant to clarithromycin,levofloxacin and metronidazole;2.Construct ce RNA network of H.pylori infection-related gastric cancer to explore the biological function and prognosis of related molecules.This study will provide clues and theoretical basis for further in-depth research on drug resistance and pathogenicity related to H.pylori infection.Methods: 1.Molecular docking simulation analysis of the interaction between 9 23 S r RNA resistance-related mutation sites,35 rdx A resistance-related mutation sites,4 gyr A resistance-related mutation sites and clarithromycin,levofloxacin,and metronidazole,to further identify possible resistance-associated mutation sites.Further,use natural transformation methods for in vitro validation of selected sites.2.Extracting RNA expression data from TCGA database with or without H.pylori in gastric cancer patients,including 20 patients with H.pylori-infected gastric cancer and 168 patients without H.pylori gastric cancer.Seventy patients with fresh gastric cancer tissues who underwent gastric cancer resection between 2012 and 2014 were selected.We used R software to calculate differentially expressed lnc RNA,mi RNA and m RNA,construct lnc RNA-mi RNA-m RNA ce RNA network,and performed GO and pathway enrichment analysis on differentially expressed m RNA.Besides,we constructed a PPI interaction network through the STRING database to detect differential expression Lnc RNA and mi RNA were used for survival analysis.Finally,gastric cancer tissue samples were used for q RT-PCR verification.Results: 1.Through molecular docking analysis,the binding of H.pylori resistance-associated sites A2143 G,G1949A,C1953 T,G2211T and CAM weakened after mutation.After further natural transformation verification,G1949 A was a new mutation site related to CAM resistance,and double-mutant strains(G1949A and A2143G)on 23 S could grow on plates with higher CAM concentration.Besides,a total of 24 mutation sites with weaker binding to MNZ were found,and the natural transformation test successfully verified the stop codon mutation of 110 site and point-shift mutation of 58 site and M21 A mutation on the rdx A gene were closely related to MNZ resistance.By molecular docking analysis,the mutation sites of gyr A associated with LVX resistant were screened mainly at 87,91,99,and 143.2.By using TCGA database for bioinformatics analysis,we found 27 DElnc RNA,32 DEmi RNA and 257 DEm RNA in the comparison of GC with or without H.pylori infection.These genes might be involved in the regulation of GC development by H.pylori infection.Finally,11 DElnc RNAs,10 DEmi RNAs and 219 DEm RNAs were screened to construct the ce RNA network of H.pylori-positive GC.Secondly,the cellular functions and signaling pathways of DEm RNAs involved in the ce RNA network were predicted.GO analysis showed that sequence-specific DNA binding,proteolysis and exosomes were the most significant,and the PI3K-Akt signaling pathway was the most likely pathway.The most meaningful functional module was extracted from the protein interaction analysis.It may be involved in the process of keratin filaments and keratinization to affect the development of the disease.The relationship between total survival rate and DElnc RNA and DEmi RNA in the ce RNA network was explored.From the results of survival analysis,there were 4 DElnc RNA(LINC01254,LINC01287,LINC01524,U95743.1)and four DEmi RNAs(mi R-302 b,mi R-302 a,mi R-378 g,mi R-1286)related to the survival of patients with H.pylori-positive GC,which might be potential indicators for prognosis.Finally,we used the q RT-PCR validation further validated our bioinformatics analysis results.The results showed that the expressions of LINC01254,LINC01287,LINC01524,U95743.1 in H.pylori-positive GC tissues were higher than in H.pylori-negative GC tissues,which were consistent with the predicted results of TCGA.Conclusion: 1.In this study,through molecular docking simulation analysis of H.pylori gene mutation sites found in previous studies that were related to CAM,MNZ,and LVX resistance,respectively.A2143 G,G1949A,C1953 T and G2211 T mutations on 23 s r RNA related to CAM resistance;24 new mutation sites on the rdx A gene associated with MNZ resistance and mutations at 87,91,99,143 sites on the gyr A gene associated with LVX resistance were screened out.The natural transformation experiment was used to verify that G1949 A was related to CAM resistance,and had a synergistic effect on A2143 G mutation resistance.It was also successfully confirmed that the 110 and 58 sites and the M21 A mutation were related to MNZ resistance.2.Using the TCGA database for data mining,we successfully constructed a ce RNA regulatory network of H.pylori-infected GC,consisting of 10 lnc RNAs,11 mi RNAs,219 m RNAs,and analyzed their possible biological processes and pathways.Further screening revealed 4 DElnc RNA and 4 DEmi RNA related to GC prognosis.RT-PCR experiments confirmed that LINC01254,LINC01287,LINC01524,U95743.1 were closely related to H.pylori-positive GC.