Screening And Identification Of Small Non-coding RNAs In H.Pylori And The Negative Regulatory Role Of MiRNAs In The Inflammatory Response During H.Pylori Infection

Recently, small non-coding RNAs, which are a group of regulatory RNA molecules normally without a protein-coding function, have attracted great interest as key regulators in both eukaryotic and prokaryotic life.In bacteria, these regulatory RNAs are also termed as small RNAs or sRNAs. The majority of sRNAs can control gene expression at the post-transcriptional level via base pairing with complementary sequences in target transcripts. Many sRNAs have been identified as crucial regulatory elements in bacterial stress responses, bacterial virulence, quorum sensing and bacterial homeostasis. As a result of recent systematic searches, over 70 sRNAs are now known in Escherichia coli, and more and more sRNAs are found in other pathogens, for example, Salmonella, Yersinia and Vibrio harveyi, etc.microRNAs (miRNAs) are small noncoding RNAs that posttranscriptionally regulate gene expression. Mature miRNAs can specifically bind to 3′UTRs of target cellular mRNA in turn triggering mRNA degradation or inhibition of translation. miRNAs can act as key regulators in a wide variety of biological processes and various diseases, including development, cell differentiation, apoptosis, metabolism, and cancer.Helicobacter pylori (H. pylori) is a major human pathogen which is associated with gastric diseases like chronic active gastritis, peptic ulcer, and gastric carcinoma. A remarkable feature of H. pylori is its ability to survive in the extremely acidic environment of the stomach. It requires regulation of bacterial gene expression to cope with the environmental fluctuations. Despite the limited number of transcriptional regulators, evidence is accumulating for the existence of new and complex circuits regulating gene transcription. Another remarkable feature of the H. pylori infection is its complex immune response. Though strong cellular and humoral immunity is induced during H. pylori infection, the immune and inflammatory response is unable to clear the bacterium, resulting in lifelong bacterial persistence. During its long co-existence with humans, H. pylori, host and environmental factors consist of a complex network to precisely regulate the immune response, however, the regulatory mechanism of this complex system is not clear.To date, little is known about sRNAs and H. pylori, and the potential role of miRNAs in the immune response to H. pylori infection has not been investigated. Here we first systematically identified sRNAs in H. pylori genome by a computational approach and a novel experimental strategy based on RNase I protection assay. The expression profile of cellular miRNAs during H. pylori infection was analyzed by using microarray, quantitative RT-PCR and Northern blot. Then we choose miR-155 for detailed investigation, defined the underlying mechanism leading to the miR-155 up-regulation by H. pylori, and identified the potential target genes of miR-155, and investigated the possible role of miR-155 as novel negative regulator that help to fine-tune the inflammation response of H. pylori infection. Our results provide insight into the knowledge of regulatory network of H. pylori. Furthermore, the altered miR-155 expression may identify a potential link between miRNAs and immune regulation during H. pylori infection.Methods1. Identification of sRNAs in H. pylori by a bioinformatics based approach. A file of all H. pylori Intergenic regions (IGRs) sequences was created by a Perl package based on the gene annotations. Then we systematically identified sRNAs in H. pylori based on gene location, sequence conservation, promoter and terminator search, and secondary structure. The candidate sRNAs were confirmed by Northern blot and RT-PCR.2. Screening of natural antisense transcripts(NATs) in H. pylori by a novel approach based on RNase I protection assay. Total RNA H. pylori was digested by RNase I to remove single-stranded RNA. Synthesis of the first strand of cDNA was performed with random primer and reverse transcriptase. Then cDNA was tailed with poly(A) at the 3′-end, and a 5′adapter was ligated to 5' phosphorylated cDNA, and a cDNA library of NATs was constructed by PCR.3. Identification of NATs in H. pylori. After the identification by SmaⅠ/XhoⅠrestriction endonuclease, insert-containing clones were subsequently characterized by DNA sequencing and BLAST analysis. The expression of NATs was confirmed by Northern blot and poly(A)-tailed RT-PCR.4. The expression profile of miRNAs in gastric epithelial cell lines and gastric mucosal tissues during H. pylori infection was analyzed by using microarray, quantitative RT-PCR and Northern blot.5. The negative regulatory role of miR-155 in the inflammatory response.The potential target of miR-155 was identified by luciferase assay and western blot. Promoter analysis and inhibitor experiment were used to investigate the pathway involved in the induction of miR-155. Examination of miR-155 function was performed by overexpression and inhibition of miR-155.Results:1. Identification of sRNAs in H. pylori by a bioinformatics based approach.Among a total of 6 candidate sRNAs initially predicted, two novel sRNAs (IG-443 and IG-524) were confirmed by Northern blot and RT-PCR. Virtually, they were a class of natural antisense transcripts (NATs), which were complementary to partial sequences of the following genes: flagellar motor switch gene (fliM) and fumarase (fumC).2. Screening and identification of NATs in H. pylori by a novel approach.We successfully constructed a cDNA library of NATs containing 68 clones. After the identification by SmaⅠ/XhoⅠrestriction endonuclease, a total of 33 insert-containing clones were subsequently characterized by DNA sequencing and BLAST analysis. 6 of all inserts were partially homologous to H. pylori genomic sequences and corresponded to the opposite strands of ORFs. Two novel NATs (NAT-39 and NAT-67) were confirmed by Northern blot and poly(A)-tailed RT-PCR. They were respectively complementary to the following genes: iron-regulated outer membrane protein (frpB) and periplasmic iron-binding protein (ceuE). NAT-39 and NAT-67 may participate in the regulation of iron homeostasis in H. pylori in a sequence complementary manner.3. Screening and identification of obviously altered miRNAs in response to H. pylori infection.The expression of miRNAs could be significantly altered during H. pylori infection, including the up-regulation of miR-155, miR-16, and miR-146a, and the down-regulation of miR-181b and miR-324. Consistent with the microarray findings, the results of qRT-PCR showed that miR-155, miR-16, and miR-146a was increased, 3.0, 2.1 and 2.5 fold change, respectively. miR-155 was highly up-regulated in H.pylori-positive patients, with a 3.9-fold increase as compared to the control (P<0.05). 4. The 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-155.5. IKK-ε, SMAD2, and FADD are potential targets of miR-155, and miR-155 might down-regulate the target protein through different mechanism, either mRNA degradation or inhibition of translation.6. miR-155 mimics significantly attenuated the mRNA and protein levels of IL-8 and GRO-α, 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.Conclusion:1. We developed a computational approach to identify novel sRNAs in H. pylori, and identified two sRNAs (IG-443 and IG-524). The results indicate that there exist novel sRNAs in H. pylori and these RNAs may play potential role in regulating gene expression.2. We developed a novel approach based on RNase I protection assay to identify NATs in H. pylori, and identified two NATs (NAT-39 and NAT-67). They may participate in the regulation of iron homeostasis in H. pylori in a sequence complementary manner.3. H. pylori can stimulate the expression of miR-155 in gastric epithelial cells as well as in gastric mucosal tissues. miR-155 may function as novel negative regulator that help to fine-tune the inflammation response of H. pylori infection. Furthermore, the altered miR-155 expression may identify a potential link between miRNAs and pathogenesis of H. pylori related diseases.