Identification And Functional Analysis Of Non-coding RNAs Involved In Helicobacter Pylori-associated Infection

Helicobacter pylori（H.pylori）is one of the human gastric pathogens which infectsmore than 60% of the world’s population.The complicated pathogenic potential of H.pylori is driven by the interplay between bacterial virulence factors and the host’s immune responses which resulting in persistent gastric mucosal damages.We are still far from unveiling the exact regulatory mechanism of this complex mechanism.In recent years,studies have shown that non-coding RNAs（nc RNAs）,mainly micro RNAs（mi RNAs）and long non-coding RNAs（lnc RNAs）,play a role as regulatory molecules in the regulation of many biological processes,diseases and tumors.However,pathogenic functions of nc RNAs involved in H.pylori infection are still currently unknown or unclear.In this research,we screened and identified the profile of altered nc RNAs in H.pylori-infected gastric epithelial cells and tissues by microarrays.We concentrated on hsa-mir-29a-3p,which overexpressed in H.pylori-infected gastric epithelial cells and tissues.Bioinformatics,molecular biology,cell biology and immunology techniques were used for exploring the function of hsa-mir-29a-3p.This research may shed light on the pathogenesis of H.pylori infection.Methods:1.H.pylori 26695 strains were co-cultured with gastric epithelial cells GES-1 at a multiplicity of infection（MOI）of 100;the expression levels of IL-8 and Cox-2 were analysed by ELISA assay and Western blot respectively.2.Microarray analysis was employed to explore the m RNAs and nc RNAs expression profiles in H.pylori infected groups and control groups.Distinguishable nc RNAs and m RNAs were studied by Hierarchical Clustering analysis.3.MRNAs-nc RNAs co-expression network was built according to the normalized signal intensity of specific expression in nc RNAs and m RNA based on Pearson correlation coefficient method.Gene Ontology（GO）and KEGG Pathway enrichment analysis were performed to clarify the functions and biological pathways of differentially expressed nc RNAs co-expressed m RNAs from microarray data.4.The differentially expressed candidate nc RNAs in different infection models were verified by q RT-PCR system.VI5.Nc RNAs expression patterns in clinical specimens were identified via q RT-PCRsystem;and one-way ANOVA with post hoc method were included for multiple group comparisons.6.Differentially expressed hsa-mir-29a-3p was detected by q RT-PCR system in different infection models and clinical specimens.7.Transient transfection of GES-1 cells with hsa-mir-29a-3p mimic/inhibitor;the signal transduction,inflammation,and tumor formation-associated proteins were detected by Western blot.8.The potential target of hsa-mir-29a-3p was predicted by bioinformatical methods.The dual-luciferase reporter vector containing m RNA 3’-UTR or its mutation were constructed.The Dual-luciferase reporter assay system was applied for compare the activity of luciferase in HEK-293 T cells.9.To explore the interaction of hsa-mir-29a-3p and its target,the target protein levels modulated by hsa-mir-29a-3p in GES-1 cells as well as during H.pylori infection were determined by Western blot analysis.10.Western blot analysis was used to detect the expression of target protein in different infection models.QRT-PCR and immunohistochemistry assay were used to detect the gene and protein expression level in H.pylori-positive and negative clinical specimens.11.Western blot,immunofluorescence and ELISA assay were applied to detect the regulation effect of A20 in gastric epithelial cells and H.pylori-triggered inflammation.Results:1.Five hundred and sixty-five m RNAs and 347 nc RNAs were identified as aberrantly expressed transcripts（≥2 or ≤0.5-fold change,P<0.05）in cell models response to H.pylori infection compared with controls.2.MRNAs-nc RNAs co-expression network was built and displayed that nc RNAs/m RNAs were significantly correlated with their adjacent m RNAs/nc RNAs.GO and KEGG Pathway enrichment analysis showed that the most enriched GO terms and pathways targeted by aberrantly regulated m RNAs were involved in cell proliferation and differentiation,metastasis,apoptosis,immune responses,and gene transcription.3.Altered-expressed includingnc RNAs,n345630,XLOC₀04787,n378726,LINC00473,hsa-mir-4500,hsa-mir-and29 a hsa-mir-beenhad221,defined in H.pylori-infected groups using microarray analysis.4.Down-regulated XLOC₀04787,LINC00473 showed significant correlations with H.pylori positive chronic inflammation and intestinal metaplasia;while up-regulated hsa-mir-29 a and hsa-mir-221 showed significant correlations with H.pylori positive chronic gastritis;hsa-mir-221 showed significant correlations with H.pylori positive gastric cancer.5.A20 is a direct target of hsa-mir-29a-3p,which was harbored hsa-mir-29a-3p seed region in its 3’UTR.Protein levels of A20 were strongly reduced on the introduction of hsa-mir-29 a mimic into GES-1 cells,and also into H.pylori infected GES-1 cells.6.The target A20 was involved in regulation of NF-κB pathway activation in gastric epithelial cells.Conclusions:1.Firstly used HTA 2.0 microarray to detect the expression profiles of nc RNAs in H.pylori-infected GES-1 cells in vitro.The dysregulated modulation RNA molecules may provide new strategies for studying the pathogenesis of H.pylori infection in future.2.GO and KEGG Pathway enrichment analysis offered us a reliable way to point out the candidate biological functions and pathways that the nc RNAs interacted m RNAs involved in.Presuming that the aberrantly expressed nc RNAs which were co-expressed with those genes in network may have involvements in and play collective roles in mediating such processes in H.pylori infection.3.A subset of aberrantly expressed nc RNAs in H.pylori-infected models and gastric mucosa tissues were identified,those which might contribute to the pathological processes during H.pylori infection.4.Up-regulated hsa-mir-29a-3p may negatively contribute to H.pylori-induced inflammation by regulation of its target A20.