**6. Cloning the MG** *COX-2* **promoter region**

As mentioned above, human *COX-2* has abundant CGIs in the promoter region. To examine whether CGIs are present in the *COX-2* promoter region of MGs, we performed genomic PCR with primers designed using as previously reported MG cDNA (Accession #: AB177842) [33]. The PCR product has an intron sequence in the promoter region. Then, gene walking was performed using a primer in this intron. The promoter region of the MG *COX-2* gene was obtained (**Figure 6**), and the sequence contains more CGIs than the mouse genome, and is comparable to that of the human genome. Pyrosequencing showed several methylated CGIs (>15%) in the MGC2 cells. Treatment with 5-aza-dC decreased the methylation levels in these cells (**Figure 7**). As was observed in the human stomach, *COX-2* methylation levels were increased in *H. pylori*-infected gastric mucosa of MGs when compared to the levels in

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**Figure 7.** Effect of 5-aza-dC treatment on *COX-2* methylation levels in MGC2 cells. Cells were treated with vehicle (A) with 5-aza-dC (B, 1 μmol/L) for 5 days, and then methylation levels of *COX-2* were analyzed by quantitative bisulfitepyrosequencing methods as previously reported [42]. Bisulfite treatment of gDNA was performed with the EpiTect bisulfite kit (QIAGEN, Valencia, CA, USA) according to the manufacturer's protocol. Bisulfite-treated DNA (1 μL) was used as a template in subsequent PCR experiments. For most assays, we used touchdown PCR. All PCR assays included a denaturation step at 95°C for 30 s, followed by annealing at various temperatures for 30 s, and extension at 72°C for 30 s. After PCR, the biotinylated strand was captured on streptavidin-coated beads (Amersham Bioscience, Uppsala, Sweden) and incubated with sequencing primers. Pyrosequencing was performed using PSQ HS 96 Gold single-nucleotide polymorphism reagents on a PyroMark Q24 pyrosequencing machine (QIAGEN). The protocol for pyrosequencing was described in detail previously [42]. Pyrosequencing quantitatively measures the methylation status of CpG sites in a target region. Adjacent sites usually show highly concordant methylation. Therefore, the mean percent methylation in detected sites can be used as a representative value for each gene promoter. Cases with the methylation density > 15% were regarded as methylation positive. Forward primer: tgggtgaggggaattttataga, reverse primer: aaaccctaaccatccttacaa;

the control mucosa (**Figure 8**).

and sequencing primer: aggagtttgtttaggaag.

**Figure 6.** Human, MG, and mouse *COX-2* promoter regions. Genome walking to isolate genomic, the *COX-2* promoter region of MGs, was performed by using the Straight Walk Kit [41] according to the manufacturer's instructions (Bex, Tokyo, Japan). Amplified fragments were cloned into the pSTBlue-1 vector (Novagen, Madison, WI, USA) and sequenced. More abundant CGIs were observed in the MG genome when compared to the mouse genome, and the number was comparable to human genome.

is comparable to that of the human genome. Pyrosequencing showed several methylated CGIs (>15%) in the MGC2 cells. Treatment with 5-aza-dC decreased the methylation levels in these cells (**Figure 7**). As was observed in the human stomach, *COX-2* methylation levels were increased in *H. pylori*-infected gastric mucosa of MGs when compared to the levels in the control mucosa (**Figure 8**).

PCR with primers designed using as previously reported MG cDNA (Accession #: AB177842) [33]. The PCR product has an intron sequence in the promoter region. Then, gene walking was performed using a primer in this intron. The promoter region of the MG *COX-2* gene was obtained (**Figure 6**), and the sequence contains more CGIs than the mouse genome, and

**Figure 6.** Human, MG, and mouse *COX-2* promoter regions. Genome walking to isolate genomic, the *COX-2* promoter region of MGs, was performed by using the Straight Walk Kit [41] according to the manufacturer's instructions (Bex, Tokyo, Japan). Amplified fragments were cloned into the pSTBlue-1 vector (Novagen, Madison, WI, USA) and sequenced. More abundant CGIs were observed in the MG genome when compared to the mouse genome, and the

number was comparable to human genome.

214 Chromatin and Epigenetics

**Figure 7.** Effect of 5-aza-dC treatment on *COX-2* methylation levels in MGC2 cells. Cells were treated with vehicle (A) with 5-aza-dC (B, 1 μmol/L) for 5 days, and then methylation levels of *COX-2* were analyzed by quantitative bisulfitepyrosequencing methods as previously reported [42]. Bisulfite treatment of gDNA was performed with the EpiTect bisulfite kit (QIAGEN, Valencia, CA, USA) according to the manufacturer's protocol. Bisulfite-treated DNA (1 μL) was used as a template in subsequent PCR experiments. For most assays, we used touchdown PCR. All PCR assays included a denaturation step at 95°C for 30 s, followed by annealing at various temperatures for 30 s, and extension at 72°C for 30 s. After PCR, the biotinylated strand was captured on streptavidin-coated beads (Amersham Bioscience, Uppsala, Sweden) and incubated with sequencing primers. Pyrosequencing was performed using PSQ HS 96 Gold single-nucleotide polymorphism reagents on a PyroMark Q24 pyrosequencing machine (QIAGEN). The protocol for pyrosequencing was described in detail previously [42]. Pyrosequencing quantitatively measures the methylation status of CpG sites in a target region. Adjacent sites usually show highly concordant methylation. Therefore, the mean percent methylation in detected sites can be used as a representative value for each gene promoter. Cases with the methylation density > 15% were regarded as methylation positive. Forward primer: tgggtgaggggaattttataga, reverse primer: aaaccctaaccatccttacaa; and sequencing primer: aggagtttgtttaggaag.

*H. pylori*-infected gastric mucosa. In addition, *COX-2* promoter methylation levels in patients with successfully eradicated *H. pylori* infection are lower than the levels in *H. pylori*-positive cases. As mentioned above, experiments using rodents have shown that *COX-2* mRNA expression is enhanced during ulcer healing. Thus, we investigated the influence of *H. pylori* infection on COX-2 expression during stomach ulcer healing in MGs. Acetic acid-induced ulcer healing was delayed in *H. pylori*-infected MG stomachs when compared to the healing of ulcers without *H. pylori* infection [29]. The *COX-2* promoter region of MGs also has abundant CGIs, comparable to human *COX-2*. Thus, MGs are good models for investigating the role of *COX-2* methylation in gastric mucosal healing. While *COX-2* mRNA expression at the ulcer edge was increased 5 days after acetic acid injection in MG stomachs without *H. pylori* infection, as was observed in the mouse and rat models, such increases in *COX-2* mRNA expression were not observed in *H. pylori*-infected MG gastric mucosa. However, a demethylating agent restored COX-2 mRNA expression in both human and MG gastric carcinoma cell lines in which COX-2 is densely methylated. Accordingly, we demonstrated *in vitro* and *in vivo* that *COX-2* mRNA expression is regulated through an epigenetic mechanism in human and MG gastric mucosa. Aberrant DNA methylation has been extensively investigated in the context of the pathogenesis of various cancers, including stomach cancer. In addition, the involvement of epigenetic modifications has been reported in the pathogenesis of various chronic diseases, such as essential hypertension and cardiovascular disease [39]. Taken together, the epigenetic inhibition of COX-2 expression due to chronic inflammation induced by *H. pylori* infection seems to interfere with ulcer healing and increase the vulnerability of the gastric mucosa. The decrease in *COX-2* methylation levels after *H. pylori* eradication may restore

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*H. pylori* infection, the leading cause of peptic ulcer disease, induces sustained inflammation in the gastric mucosa. This chronic inflammation causes aberrant DNA methylation in various genes. The *COX-2* promoter regions in both MGs and humans contain abundant CGIs. COX plays critical roles in peptic ulcer development and healing, and both COX-1 and COX-2 are necessary for the gastric mucosal defense system. COX-1 is a housekeeping enzyme, which maintains microcirculation and mucous production. During ulcer healing, COX-2 is expressed at the margin of an ulcer, and COX-2-derived PGs induce various growth factors to promote mucosal healing and angiogenesis. Ulcer healing is delayed in *H. pylori* infected-MG stomachs, which is the best model of *H. pylori* infection in humans. We showed that *COX-2* mRNA induction during ulcer healing was impaired in *H. pylori*-infected-MG stomachs. The *COX-2* promoter region is methylated in both *H. pylori*-infected human and MG gastric mucosa. These CGIs are also methylated in human Kato III cells and MGC2 gastric carcinoma cells. *COX-2* mRNA expression in these cells is restored by treatment with a demethylating agent. In *H. pylori*-infected gastric mucosa, *COX-2* promoter methylation appears to be involved in the impaired *COX-2* mRNA induction typically observed during ulcer healing,

gastric mucosal defense.

which leads to delayed ulcer healing.

**8. Conclusions**

**Figure 8.** DNA methylation levels at *COX-2* in the MG gastric mucosa in the presence or absences of *H. pylori* infection. Methylation levels at *COX-2* in the MG gastric mucosa were analyzed by quantitative bisulfite pyrosequencing.
