J Gastric Cancer.  2013 Dec;13(4):232-241.

Gastrokine 1 Expression in the Human Gastric Mucosa Is Closely Associated with the Degree of Gastritis and DNA Methylation

• Choi WS
• Seo HS
• Song KY
• Yoon JH
• Kim O
• Nam SW
• Lee JY
• Park WS

Abstract

PURPOSE
Gastrokine 1 plays an important role in gastric mucosal defense. Additionally, the Gastrokine 1-miR-185-DNMT1 axis has been shown to suppress gastric carcinogenesis through regulation of epigenetic alteration. Here, we investigated the effects of Gastrokine 1 on DNA methylation and gastritis.
MATERIALS AND METHODS
Expression of Gastrokine 1, DNMT1, EZH2, and c-Myc proteins, and the presence of Helicobacter pylori CagA protein were determined in 55 non-neoplastic gastric mucosal tissue samples by western blot analysis. The CpG island methylation phenotype was also examined using six markers (p16, hMLH1, CDH1, MINT1, MINT2 and MINT31) by methylation-specific polymerase chain reaction. Histological gastritis was assessed according to the updated Sydney classification system.
RESULTS
Reduced Gastrokine 1 expression was found in 20 of the 55 (36.4%) gastric mucosal tissue samples and was closely associated with miR-185 expression. The Gastrokine 1 expression level was inversely correlated with that of DNMT1, EZH2, and c-Myc, and closely associated with the degree of gastritis. The H. pylori CagA protein was detected in 26 of the 55 (47.3%) gastric mucosal tissues and was positively associated with the expression of DNMT1, EZH2, and c-Myc. In addition, 30 (54.5%) and 23 (41.9%) of the gastric mucosal tissues could be classified as CpG island methylation phenotype-low and CpG island methylation phenotype-high, respectively. Reduced expression of Gastrokine 1 and miR-185, and increased expression of DNMT1, EZH2, and c-Myc were detected in the CpG island methylation phenotype-high gastric mucosa.
CONCLUSIONS
Gastrokine 1 has a crucial role in gastric inflammation and DNA methylation in gastric mucosa.

Keyword

Gastrokine 1; DNA methylation; microRNA-185; CagA protein; Gastric mucosa

MeSH Terms

Axis, Cervical Vertebra
Blotting, Western
Carcinogenesis
Classification
CpG Islands
DNA Methylation*
DNA*
Epigenomics
Gastric Mucosa*
Gastritis*
Helicobacter pylori
Humans*
Inflammation
Methylation
Mucous Membrane
Phenotype
Polymerase Chain Reaction
Proto-Oncogene Proteins c-myc
DNA
Proto-Oncogene Proteins c-myc

Figure

• 

  Fig. 1 GKN1 protein is a representative marker of the degree of gastritis. (A) Western blotting analysis with GKN1, CagA, DNMT1, EZH2, and c-Myc in gastric mucosal tissues. (B) Based on scatterplot analysis, GKN1 expression was closely associated with mononuclear cell infiltration (Student's t-test, P=0.0332), atrophy (P<0.0001), and IM (P=0.0079), but not with granulocytic infiltration (P=0.4889). (C) ROC curve analysis using GKN1 to distinguish patients with atrophy and intestinal metaplasia from human gastric mucosal tissues. GKN1 yielded an AUC value of 0.865 (95% confidence interval [CI], 0746~0.942; P<0.0001) and 0.973 (95% CI, 0889~0.998; P<0.0001) in distinguishing atrophy and intestinal metaplasia, respectively. GKN1 = Gastrokine 1; DNMT1 = DNA methyltransferase 1; EZH2 = enhancer of zeste homolog 2; ROC = receiver operating characteristic; AUC = area under the ROC curve; IM = intestinal metaplasia.

• 

  Fig. 2 Expression of GKN1 is positively related to miR-185 expression and inversely related to expression of DNMT1, EZH2, and c-Myc. (A) Based on scatterplot analysis, expression of miR-185 (Pearson test, P=0.0001) was higher, while expression of DNMT1 (P<0.0001), EZH2 (P<0.0001), and c-Myc (P<0.0001) was lower in GKN1-positive (GKN1+) gastric mucosa. (B) Scatterplot analysis showed that expression of GKN1 (Pearson test, P=0.0013) and miR-185 (P=0.0014) was reduced and expression of DNMT1 (P=0.0002), EZH2 (P=0.0016), and c-Myc (P=0.0366) was increased in CagA-positive (CagA+) gastric mucosa. (C) Expression of GKN1 was positively correlated to miR-185 and negatively correlated to DNMT1, EZH2, and c-Myc expression (Pearson test, P<0.0001). The value of miR-185 was obtained by quantitative real-time RT-PCR. GKN1 = Gastrokine 1; DNMT 1 = DNA methyltransferase 1; EZH2 = enhancer of zeste homolog 2; RT-PCR = reverse transcription polymerase chain reaction.

• 

  Fig. 3 Expression levels of the epigenetic reuglators were closely associated with methylation status in gastric mucosa. (A) In the chart showing methylation in the CIMP panel (p16, MINT1, MINT2, MINT31, CDH1, and MLH1), the gray-colored boxes indicate hypermethylation. (B) Scatterplot analysis with CIMP-L and CIMP-H revealed that methylation status was closely associated with expression levels of GKN1 (Stduent's t-test, P<0.0001), miR-185 (P<0.0001), DNMT1 (P=0.0004), EZH2 (P<0.0001), and c-Myc (P<0.0001). CIMP = CpG island methylation phenotype; H = high; L = low; GKN1 = Gastrokine 1; DNMT 1 = DNA methyltransferase 1; EZH2 = enhancer of zeste homolog 2.

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