Screening And Identification Of H.Pylori-Related Non Coding RNA And Exploring Their Function

About 50% of the world’s population is infected with Helicobacter pylori（H.pylori）, which is a causative agent for gastritis, peptic ulcer disease, mucosa-associated lymphoid tissue（MALT） lymphoma, and gastric cancer. It is estimated that individuals infected with H. pylori have more than two-fold increased risk of developing gastric cancer compared with non-infected ones（Brenner et al., 2000） and nearly all gastric cancer is related to H.pylori. Why only 1 to 5% of H. pylori infected persons develop gastric cancer remains unknown and it seems to depend on the relationship between environmental, bacterial virulence factors and host genetics.Noncoding RNA can be divided to be microRNAs and lncRNAs in referencing of its length. MicroRNAs（miRNAs） are small noncoding RNAs that are implicated in many physiological and pathological responses 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. Long noncoding RNA（lncRNAs）, which were well known as more than 200 nt in length, are acknowledged by its variousness of the sequences and mechanisms. There has been recent evidence regarding novel relationship between miRNAs and H. pylori induced gastric carcinogenesis. Although sporadic pubilications reported that lncRNA might play important roles during cellular development and their misregulation has also been shown in various types of diseases, however, the report of potential role of lncRNAs in the immune response to pathogen is rare. In this study, we identified nonoverlapping signatures of a small number of lncRNAs and microRNAs that are aberantly expressed in H.pylori infected gastric epithelial cell via a microarray analysis followed by bioassays. And the functions of certain noncoding RNAs were further determined. Section1 Genome-wide analysis of long noncoding RNA profile in human gastric epithelial cell response to Helicobacter pylori.The GES-1 cells were infected by H. pylori. The expression profile of cellular lncRNAs during H. pylori infection was analyzed by RNA-seq followed by RT-PCR. We identified nonoverlapping signatures of a small number of lncRNAs that are aberantly expressed in H.pylori infected gastric epithelial cell via a microarray analysis followed by bioassays. From the microarray data, we found that 24 lncRNAs were up-regulated and 22 were down-regulated. Five lncRNAs, XLOC₀04562（p<0.05）, XLOC₀05912, XLOC₀00620, XLOC₀04122（ p<0.05） and XLOC₀14388（p<0.05） were further evaluated by q RT-PCR and the results matched well with microarray data. XLOC₀04122 and XLOC₀14388 were also decreased in gastric mucosal tissues of H.pylori positive patients. Differentially expressed lncRNAs might play a partial or key role in the immune response against H. pylori and this might provide potential targets for the future treatment of H. pylori related diseases. Section2 MicroRNA-146 a enhance Helicobacter pylori induced cell apoptosis in human gastric cancer epithelial cell.The micro RNA-146 a expression profile and cell apoptosis of MKN7 cell line were determined during H.pylori infection by quantitative RT-PCR and V-Fluos staining kit. Examination of micro RNA-146 a function in H. pylori infection were performed by overexpression and inhibition of micro RNA-146 a. Weather micro RNA-146 a functions to inhibit cell apoptosis by targeting COX-2 were also determined by COX-2 overexpression. Clinical data was also collected to determine the relationship between micro RNA-146 a and prognosis of gastric cancer. We found that overexpression of micro RNA-146 a by transfecting micro RNA-146 a mimics could significantly enhance apoptosis, and this up-regulation of apoptosis was triggered by COX-2 inhibition（p<0.05）. Furthermore, we found that micro RNA-146 a density was positively correlated with apoptosis rates in gastric mucosal tissues from H. pylori–positive gastric cancer patients（p<0.05）. Meanwhile, intratumoral micro RNA-146 a density was negatively correlated with lymph node metastasis（p<0.05）. Understanding the important roles of micro RNA-146 a in regulating cell apoptosis in human gastric cancer epithelial cell will contribute to the development of micro RNA- targeted therapy in the future.Section3 micro RNA-99 b promotes Helicobacter pylori-induced autophagy and suppresses carcinogenesis by targeting m TOR.The micro RNA-99 b expression profile and cell apoptosis of gastric cancer cells were determined during H.pylori infection by quantitative RT-PCR. Examination of micro RNA-99 b function of intracellular bacterial clearance and autophagy promotion in H. pylori infection were performed by overexpression and inhibition of micro RNA-99 b. The potential target of micro RNA-99 b was identified by luciferase assay and Western blot. Promoter analysis and inhibitor experiment were used to investigate the pathway involved in the induction of micro RNA-99 b. In this study, we found that micro RNA-99 b were increased in H. pylori infected gastric cancer tissues and cell lines（p<0.05）. Overexpression of micro RNA-99 b by transfecting micro RNA-99 b mimics could significantly enhance autophagy, decrease intracellular bacterial loads and block cell proliferation, and this up-regulation of autophagy was triggered by m TOR inhibition（p<0.05）. The results have indicated that the micro RNA-99 b expression level plays a key role in preventing H.pylori related gastric cancer formation and this might provide potential targets for the future treatment of H. pylori related diseases.Conclusion:Based on our data, we found that non coding RNA was important in primary immune response against H.pylori and its carcinogenesis.Our results indicated that XLOC₀04122,XLOC₀14388,micro RNA-146 a and micro RNA-99 b play a key role in regulating immunity. Up-regulation of these molecular might help to prevent the cancer formation mediated by H.pylori infection.