Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Helicobacter pylori-induced changes in miRNA expression in human gastric epithelial cells

ABSTRACT: H. pylori infection of human gastric epithelial cells (GEC) represses H,K-ATPase alpha subunit (HK-alpha) gene transcription through NF-Kappa B p50 homodimer binding to HK-alpha promoter. The bacterial cagA and slt gene products have been implicated in HK-alpha repression, which facilitates gastric H. pylori colonization and may underlie transient clinical hypochlorhydria. We hypothesized that H. pylori also regulates H,K-ATPase expression post-transcriptionally by micro-RNA interaction with HK-alpha mRNA. Our microarray experiment examined the effect of infection by wildtype H. pylori infection or a delta-cagA H. pylori mutant on miRNA expression of human gastric cells (AGS). Our results indicate that H. pylori infection up-regulates GEC miRNAs that target the HK-alpha 3' UTR and block translation, and that CagA activity is implicated in the miRNA up-regulation. Three types of samples were generated for microarray analysis: 1) AGS cells infected with wildtype H. pylori; 2) AGS cells infected with cagA-deficient H. pylori; 3) a mock-infected sample, prepared as a control by combining total RNA from uninfected AGS cells with total RNA from wildtype H. pylori, with relative eukaryotic and prokaryotic RNA amounts matching those observed in infected samples, based on proportional ribosomal peak heights quantified by Bioanalyzer. Three experimental replicates were generated for each type of treatment.

ORGANISM(S): Homo sapiens

SUBMITTER: Jeremy Barth

PROVIDER: E-GEOD-51306 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Helicobacter pylori infection promotes chronic inflammation and plethora of DNA damages including strand breaks, base alterations, point mutations, microsatellite instability and epigenetic alterations. The gastric epitheliaI cells, in order to prevent genomic instability, require an integrous DNA damage repair (DDR) machinery which, however, has been reported to be modulated by the infection. CagA is a major Hp virulence factor that deregulates host cell functions such as proliferation, apoptosis and chromosomal integrity. Its pathogenic activity is partly regulated by tyrosine phosphorylation on repeated EPIYA-motifs. Our aim was to identify putative effects of H. pylori infection and CagA protein on DDR machinery, investigating the transcriptome of AGS cells, infected with P12 H. pylori wild-type strain, as well as, its corresponding CagA knock-out and the CagA phosphorylation-defective mutant following tyrosine (EPIYA) substitution by phenylalanine (EPIFA) in the EPIYA-C motifs. Upon RNA-Sequencing on polyA-selected transcripts we performed Differential Expression Analysis, Pathway Enrichment Analysis and visualization on KEGG Pathway Maps per DDR mechanism. Key DDR components that were observed to be downregulated in a CagA-related manner were validated via Western Blot utilizing AGS and the non-cancerous GES1 cell lines. Transcriptome analysis revealed that a notable number of DDR genes were downregulated during H. pylori infection resulting to potential modulation of Base Excision Repair, Mismatch Repair and a more intricate deregulation of Nucleotide Excision Repair, Homologous Recombination and Non-Homologous End-Joining. CagA contributes to MUTYH, FEN1, APE1, POLD1 and LIG1 downregulation and those observations were verified on the protein expression level, with the exception of APE1 that was on contrary observed to be upregulated. Our study accentuates the role of CagA, as a significant contributor of the H. pylori infection-mediated DDR modulation, disrupting the balance between DNA damage introduction and repair thus favoring genomic instability and potentially contributing to gastric carcinogenesis.

Project description:The proteins from the Fanconi Anemia (FA) pathway of DNA repair maintain DNA replication fork integrity by preventing the unscheduled degradation of nascent DNA at regions of stalled replication forks. Here, we ask if the bacterial pathogen H. pylori exploits the fork stabilisation machinery to generate double stand breaks (DSBs) and genomic instability. Specifically, we study if the H. pylori virulence factor CagA generates host genomic DSBs through replication fork destabilisation and collapse. An inducible gastric cancer model was used to examine global CagA-dependent transcriptomic and proteomic alterations, using RNA sequencing and SILAC-based mass spectrometry, respectively. The transcriptional alterations were confirmed in gastric cancer cell lines infected with H. pylori. Functional analysis was performed using chromatin fractionation, pulsed-field gel electrophoresis (PFGE), and single molecule DNA replication/repair fiber assays. We found a core set of 31 DNA repair factors including the FA genes FANCI, FANCD2, BRCA1, and BRCA2 that were downregulated following CagA expression. H. pylori infection of gastric cancer cell lines showed downregulation of the aforementioned FA genes in a CagA-dependent manner. Consistent with FA pathway downregulation, chromatin purification studies revealed impaired levels of Rad51 but higher recruitment of the nuclease MRE11 on the chromatin of CagA-expressing cells, suggesting impaired fork protection. In line with the above data, fibre assays revealed higher fork degradation, lower fork speed, daughter strands gap accumulation, and impaired re-start of replication forks in the presence of CagA, indicating compromised genome stability. By downregulating the expression of key DNA repair genes such as FANCI, FANCD2, BRCA1, and BRCA2, H. pylori CagA compromises host replication fork stability and induces DNA DSBs through fork collapse. These data unveil an intriguing example of a bacterial virulence factor that induces genomic instability by interfering with the host replication fork stabilisation machinery.

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Helicobacter pylori-induced changes in miRNA expression in human gastric epithelial cells

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