Effect of myeloid differentiation primary response gene 88 on expression profiles of genes during the development and progression of Helicobacter-induced gastric cancer

Abstract

Background: Gastric cancer is one of the most common and lethal type of cancer worldwide. Infection with Helicobacter pylori (H. pylori) is recognized as the major cause of gastric cancer. However, it remains unclear the mechanism by which Helicobacter infection leads to gastric cancer. Furthermore, the underlying molecular events involved during the progression of Helicobacter infection to gastric malignancy are not well understood. In previous studies, we demonstrated that that H. felis-infected Myd88 ^-/- mice exhibited dramatic pathology and an accelerated progression to gastric dysplasia; however, the MyD88 downstream gene targets responsible for this pathology have not been described. This study was designed to identify MyD88-dependent genes involved in the progression towards gastric cancer during the course of Helicobacter infection.

Methods: Wild type (WT) and Myd88 deficient mice (Myd88 ^-/-) were infected with H. felis for 25 and 47 weeks and global transcriptome analysis performed on gastric tissue using MouseWG-6 v2 expression BeadChips microarrays. Function and pathway enrichment analyses of statistically significant, differential expressed genes (p < 0.05) were performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) online tools.

Results: Helicobacter infection affected the transcriptional profile of more genes in Myd88 ^-/- mice compared to WT mice. Infection of Myd88 ^-/- mice resulted in the differential expression of 1,989 genes at 25 weeks (1031 up and 958 downregulated). At 47 weeks post-H.felis infection, 2,162 (1140 up and 1022 downregulated) were differentially expressed. The most significant differentially upregulated gene during Helicobacter infection in Myd88 ^-/- mice was chitinase-like 4 (chil4), which is involved in tissue remodeling and wound healing. Other highly upregulated genes in H. felis-infected Myd88 ^-/- mice included, Indoleamine 2,3-Dioxygenase 1 (Ido1), Guanylate binding protein 2 (Gbp2), ubiquitin D (Ubd), β ₂ -Microglobulin (B2m), CD74 antigen (Cd74), which have been reported to promote cancer progression by enhancing angiogenesis, proliferation, migration, metastasis, invasion, and tumorigenecity. For downregulated genes, the highly expressed genes included, ATPase H+/K+ transporting, alpha subunit (Atp4a), Atp4b, Mucin 5 AC (Muc5ac), Apolipoprotein A-1 (Apoa1), and gastric intrinsic factor (Gif), whose optimal function is important in maintaining gastric hemostasis and lower expression has been associated with increased risk of gastric carcinogenesis.

Conclusions: These results provide a global transcriptional gene profile during the development and progression of Helicobacter-induced gastric cancer. The data show that our mouse model system is useful for identifying genes involved in gastric cancer progression.

Keywords: Gastric cancer; Gene regulation; Helicobacter; Microarray; MyD88.

Fig. 1

Multi Dimensional Scale Plot of all H. felis infected and control samples from WT and Myd88 ^−/− mice. Samples were separated using 8 RNA SEQ libraries based on sample relations of 23,015 genes with a standard deviation/ mean >0.1 Groups separated into infected Myd88 ^−/−, infected WT and uninfected samples

Fig. 2

Venn diagram of differentially expressed genes. The number of changed genes following infection with H. felis in WT and Myd88 ^−/− mice at 25 and 47 weeks (p < 0.05) is shown. The relationship between these DEGS is also shown

Fig. 3

Scatterplot of Differentially Expressed Genes in H. felis- infected Myd88 ^−/− samples at (a) 25 and (b) 47 weeks. Scatterplot represents a summary of t-tests for individual genes, depicting the Log₂ fold changes and their corresponding –log₁₀ p-values of all differentially expressed genes from microarray analysis. Genes were separated into different time points. Negative values of Log₂ fold changes indicate downregulated genes. Positive Log₂ fold changes indicate upregulated genes. Genes with a fold change < 2.0 and a p value <0.05 are depicted as red dots and genes not found to be significantly altered are depicted as black dots. All infected animals were normalized to uninfected control mice at the same time point

Fig. 4

Network characterization of selected genes at 25 weeks. STRING gene networks of interactions of DEGs with a STRING interaction confidence of 0.7 or greater (high confidence) for H. felis-infected Myd88 ^−/− mice at 25 weeks for both upregulated (a) and downregulated (b) genes. Known interactions are illustrated with light blue string attachments (from curated databases) and light pink/purple strings (experimentally determined). Co-expression are illustrated with black string attachments

Fig. 5

Network characterization of selected genes at 47 weeks. STRING gene networks of interactions of DEGs with a STRING interaction confidence of 0.7 or greater (high confidence) for MyD88 ^−/− - H. felis infected at 47 weeks both upregulated (a) and downregulated (b) genes. Known interactions are illustrated with light blue string attachments (from curated databases) and light pink/purple strings (experimentally determined). Co-expression are illustrated with black string attachments

Fig. 6

Profiles of GO enrichment analysis. Enriched Go terms are shown for H. felis-infected Myd88 ^−/− mice at both 25 (a, b) and 47 weeks (c, d). Biological processes are depicted in figures A and C while molecular functions are depicted in B and D. For the biological processes, the top 20 processes are shown. All scores depicted are relative scores for number of genes in each function/ process relative to the number of total genes entered into the STRING database

Fig. 7

Venn Diagram showing distribution of different significantly enriched KEGG pathways. Pathways with p-value > E-04 are included. Antigen processing and presentation is the most enriched pathway overlapping the majority of genes with all other pathways. Notably, all processes are associated with inflammatory responses of the immune system