Screening, Identification And Function Analysis Of MicroRNAs In Helicobacter Pylori-induced Inflammatory Response

Helicobacter pylori (H. pylori) is one of the most popular bacteria, which is closely linked to the development of gastritis, peptic ulcer diseases, mucosa-associated lymphoid tissue (MALT) lymphoma and gastric cancer. H. pylori has been classified as Type I carcinogen by the World Health Organization. The remarkable feature of the H. pylori infection is its complicated immune response. Though strong cellular and humoral immunity is induced by H. pylori infection, the immune and inflammatory responses are unable to clear the bacteria, resulting in lifelong bacterial persistence. We are still far from unveiling the exact regulatory mechanism of this complex system.MicroRNAs (miRNAs) are a recently discovered class of small noncoding RNAs that are implicated in many physiological and pathological processes as post-transcriptional repressors of gene expression. Mature miRNAs can specifically bind to 3'-UTRs of target cellular mRNA in turn triggering mRNA degradation or inhibition of translation. In general, miRNAs act as key regulators in development, differentiation, homeostasis, and cancers.As the earlier reaction of the cells receiving exogeous and endogenous pressure, miRNAs are involved in modulating immune response. However, the regulatory role of miRNAs in bacteria infection and immunity is not clear. H. pylori-induced innate immune response mediated by gastric epithelium cells acts as the first line of defense against infection. Here we firstly established stable gastric epithelium cells model infected by H. pylori, and by microarray we analyzed the expression profile of cellular miRNAs during H. pylori infection. Then we chose miRNAs which expression were significantly altered for detailed investigation objects, and identified their expressions by the quantitative RT-PCR and Northern blot assays. Subsequently, we investigated the underlying mechanism leading to miRNAs differential expression by H. pylori, and identified the potential target genes of miRNAs by bioinformatics prediction and reporter vector system, and investigated the possible roles of miRNAs as novel negative regulator that help to fine-tune the inflammation response of H. pylori infection. Our results provided insights into the regulatory networks of H. pylori-induced inflammations. Moreover, the altered miRNAs expression may identify a potential link between miRNAs and immune regulation during H. pylori infection.Methods1. Screening and identification of H. pylori-induced miRNAsThe gastric epithelium cells model infected by H. pylori were established. The expression profile of cellular miRNAs during H. pylori infection was analyzed by microarray. Then miRNAs which expression were significantly altered were screened and identified in several infection models and in H. pylori-infected gastric mucosal tissues by the quantitative RT-PCR and Northern blot.2. Study on the mechanism of miRNAs differential expression in H. pylori infectionmiRNAs which expression were significantly altered were identified as the detailed investigation objects, and their alteration mechanism of miRNAs in H. pylori infection were analyzed by multi-stimulus analysis, promoter prediction, luciferase array and signal pathway inhibitors experiment.3. Studies on the fuction of miRNAs in H. pylori infectionThe potential targets of miRNAs in gastric epithelium cells were identified by bioinformatics prediction, luciferase reporter assay, GFP reporter assay, Realtime PCR and Western blot. Examination of miRNAs function in H. pylori infection were performed by overexpression and inhibition of miRNAs, RNAi, immunofluorescence .Results1. Screening and identification of H. pylori-induced miRNAsAfter the incubation of H. pylori 26695 with the gastric epithelium cells for 24h, induction of the scattering phenotype, IL-8 Release, and COX-2 protein expression in GES-1 cells were observed. The expression of miRNAs could be significantly altered during H. pylori infection by microarray, including the up-regulation of miR-155, miR-146a, miR-16, miR-92b, miR-30b and the down-regulation of miR-324 and miR-181b. In consensus with the findings from microarray, the results of Realtime-PCR showed that miR-155 and miR-146a in several infection models and in H. pylori-infected gastric mucosal tissues were significantly increased(P<0.01). Furthermore, miR-155 and miR-146a were highly up-regulated in H.pylori-positive patients, with 4.32 and 4.29 fold change respectively as compared with the control (P<0.01).2. Studies on the up-regulation mechanism and fuction for miR-155 in H. pylori infection2.1 Signal pathway for miR-155 up-regulation in H. pylori infectionThe promoter region of miR-155 contained putative NF-κB and AP-1 binding sites. The results of promoter analysis and inhibitor experiment showed that both NF-κB and AP-1 pathways are required for the up-regulation of miR-155 in response to H. pylori, and AP-1 plays a central role in the induction of miR-155.2.2 Prediction and identification of miR-155 targetsIKK-ε, SMAD2, and FADD are potential targets of miR-155, and miR-155 might down-regulate the target protein through mRNA degradation or translation inhibition.2.3 Inhibition of proinflammatory cytokines of miR-155 in H. pylori infectionmiR-155 mimics significantly attenuated the mRNA and protein levels of IL-8 and GRO-α(P<0.05), and the effect of miR-155 in modulating the inflammation may be as a secondary effect through diminishing NF-κB activity. miR-155 may be involved in the negative feedback regulation of inflammation.3. Studies on the up-regulation mechanism and fuction for miR-146a in H. pylori infection3.1 Signal pathway for miR-146a up-regulation in H. pylori infectionMulti-stimulus analysis showed H. pylori-related proinflammatory cytokines were not necessary to miR-146a up-regulation in H. pylori infection. Moreover, the promoter region of miR-146a contained several putative NF-κB binding sites. The results of promoter analysis and inhibitor experiment showed that NF-κB pathway is required for the up-regulation of miR-146a in response to H. pylori.3.2. Prediction and identification of miR-146a targetsIRAK1,TRAF6 and COX-2 are potential targets of miR-146a, and miR-146a might down-regulate the target protein through mRNA degradation.3.3. Inhibition of COX-2 and proinflammatory cytokines of miR-146a in H. pylori infectionmiR-146a mimics significantly attenuated the mRNA and protein levels of IL-8 , MIP-3αand GRO-α(P<0.05), and the effect of miR-146a in modulating the inflammation may be as a secondary effect through diminishing NF-κB activity. miR-146a may be involved in the negative feedback regulation of inflammation.Conclusions1. We established the gastric epithelium cells model infected by H. pylori, and analyzed the expression profile of cellular miRNAs during H. pylori infection by microarray. Then we chose miR-155 and miR-146a for detailed investigation, and identified the up-regulation in several infection cell models and in H. pylori-infected gastric mucosal tissues by the quantitative RT-PCR and Northern blot. miR-155 and miR-146a expression were positive correlated.2. Bioinformatics anaylsis indicated the promoter regions of miR-155 and miR-146a contained putative NF-κB and/or AP-1 binding sites. The results of promoter analysis and inhibitor experiment showed that NF-κB and/or AP-1 pathway were required for the up-regulation of miR-155 and miR-146a in response to H. pylori.3. As the targets of miR-155 and miR-146a, IKK-ε, SMAD2, FADD, IRAK1, TRAF6 and COX-2 were identified. miR-155 and miR-146a could significantly attenuate the mRNA and protein levels of proinflammatory cytokines induced by H. pylori infection, and the effects of miR-155 and miR-146a in modulating the inflammation may be as a secondary effect through diminishing NF-κB activity.4. miR-155 and miR-146a may function as novel negative regulators. Together with its targets, miR-155 and miR-146a may be involved in the negative feedback regulation in inflammation response of H. pylori infection. Furthermore, the altered miR-155 and miR-146a expression may establish a potential link between miRNAs and pathogenesis of H. pylori related diseases.