Autophagy-related genes in Helicobacter pylori infection

Abstract

Background: In vitro studies have shown that Helicobacter pylori (H. pylori) infection induces autophagy in gastric epithelial cells. However, prolonged exposure to H. pylori reduces autophagy by preventing maturation of the autolysosome. The alterations of the autophagy-related genes in H. pylori infection are not yet fully understood.

Materials and methods: We analyzed autophagy-related gene expression in H. pylori-infected gastric mucosa compared with uninfected gastric mucosa obtained from 136 Bhutanese volunteers with mild dyspeptic symptoms. We also studied single nucleotide polymorphisms (SNPs) of autophagy-related gene in 283 Bhutanese participants to identify the influence on susceptibility to H. pylori infection.

Results: Microarray analysis of 226 autophagy-related genes showed that 16 genes were upregulated (7%) and nine were downregulated (4%). We used quantitative reverse transcriptase polymerase chain reaction to measure mRNA levels of the downregulated genes (ATG16L1, ATG5, ATG4D, and ATG9A) that were core molecules of autophagy. ATG16L1 and ATG5 mRNA levels in H. pylori-positive specimens (n=86) were significantly less than those in H. pylori-negative specimens (n=50). ATG16L1 mRNA levels were inversely related to H. pylori density. We also compared SNPs of ATG16L1 (rs2241880) among 206 H. pylori-positive and 77 H. pylori-negative subjects. The odds ratio for the presence of H. pylori in the GG genotype was 0.40 (95% CI: 0.18-0.91) relative to the AA/AG genotypes.

Conclusions: Autophagy-related gene expression profiling using high-throughput microarray analysis indicated that downregulation of core autophagy machinery genes may depress autophagy functions and possibly provide a better intracellular habit for H. pylori in gastric epithelial cells.

Keywords: Helicobacter pylori; ATG16L1; ATG5; autophagy; single nucleotide polymorphisms.

Figure 1

Flowchart of microarray analysis. Two hundred twenty-six autophagy-related genes in Human Autophagy Database (HADb; http://autophagy.lu/) were analyzed using microarray data (compared 4 H. pylori positive and 4 H. pylori negative). Differences in autophagy-related genes expression were considered significant if the fold change of expression levels was >1.5 and the p value was <0.01.

Figure 2

Autophagy-related genes mRNA levels in the gastric specimens (50 H. pylori negative and 86 H. pylori positive). Down-regulated core autophagy-related genes (A, ATG16L1; B, ATG5; C, ATG9A; D, ATG4D) derived from microarray data were validated by qRT-PCR. Down-regulation of ATG16L1 and ATG5 mRNA levels were confirmed respectively. Beta-actin (ACTB) was used as the endogenous control for data normalization. Data were expressed by box plotting. HP,Helicobacter pylori; FC, fold change.

Figure 3

The association between ATG16L1 and ATG5 mRNA levels and H. pylori density. The ATG16L1 mRNA levels were decreased in a step-like manner by H. pylori density (Score 0, median 2.33, range 0.281–9.41; Score 1, median 1.99, range 0.997–3.03; score 2, median 1.63, range 0.135–3.12; score 3, median 1.39, range 0.105–4.17). There was no significant difference in ATG5 mRNA levels in relation to the H. pylori density score. H. pylori density were scored using the updated Sydney System. Beta-actin (ACTB) was used as the endogenous control for data normalization. Data were expressed by box plotting. FC, fold change.

Figure 4

ATG16L1 and ATG5 mRNA levels in gastric epithelial cells in response to H. pylori infection. A, ATG16L1 mRNA levels in MKN45 cells. B, ATG5 mRNA levels in MKN45 cells. C, ATG16L1 mRNA levels in MKN28 cells. D, ATG5 mRNA levels in MKN28 cells. Cells were infected with H. pylori strain 26695. ATG16L1 and ATG5 mRNA levels in H. pylori-infected and non-infected MKN45 and MKN28 cells were measured at MOI 50 and 100 for 3, 6 and 24 h. Error bars represent the standard deviation of values obtained from three experiments. Statistical differences between treated and control samples were analyzed using Student's t-test. * p < 0.05.