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MDGA2 is a novel tumour suppressor cooperating with DMAP1 in gastric cancer and is associated with disease outcome

View Article: PubMed Central - PubMed

ABSTRACT

Background: Using the promoter methylation assay, we have shown that MDGA2 (MAM domain containing glycosylphosphatidylinositol anchor 2) is preferentially methylated in gastric cancer. We analysed its biological effects and prognostic significance in gastric cancer.

Methods: MDGA2 methylation status was evaluated by combined bisulfite restriction analysis and bisulfite genomic sequencing. The effects of MDGA2 re-expression or knockdown on cell proliferation, apoptosis and the cell cycle were determined. MDGA2 interacting protein was identified by mass spectrometry and MDGA2-related cancer pathways by reporter activity and PCR array analyses. The clinical impact of MDGA2 was assessed in 218 patients with gastric cancer.

Results: MDGA2 was commonly silenced in gastric cancer cells (10/11) and primary gastric cancers due to promoter hypermethylation. MDGA2 significantly inhibited cell proliferation by causing G1–S cell cycle arrest and inducing cell apoptosis in vitro, and suppressed xenograft tumour growth in both subcutaneous and orthotopic xenograft mouse models (both p<0.001). The anti-tumorigenic effect of MDGA2 was mediated through direct stabilising of DNA methyltransferase 1 associated protein 1 (DMAP1), which played a tumour suppressive role in gastric cancer. This interaction activated their downstream key elements of p53/p21 signalling cascades. Moreover, promoter methylation of MDGA2 was detected in 62.4% (136/218) of gastric cancers. Multivariate analysis showed that patients with MDGA2 hypermethylation had a significantly decreased survival (p=0.005). Kaplan–Meier survival curves showed that MDGA2 hypermethylation was significantly associated with shortened survival in patients with early gastric cancer.

Conclusions: MDGA2 is a critical tumour suppressor in gastric carcinogenesis; its hypermethylation is an independent prognostic factor in patients with gastric cancer.

No MeSH data available.


Related in: MedlinePlus

MDGA2 is inactivated by promoter methylation in gastric cancer cell lines. (A) MDGA2 was silenced or downregulated in 10 out of 11 gastric cancer cell lines but readily expression in normal gastric tissue. The methylation status of MDGA2 was determined by methylation-specific PCR (MSP). M, methylated; U, unmethylated. (B) A typical CpG island is present at the promoter region of MDGA2. Each vertical bar represents a single CpG site. The transcription start site (TSS) is indicated by a curved arrow. A region for bisulfite genomic sequencing (BGS) and combined bisulfite restriction analysis (COBRA) is denoted. BGS analysis confirmed high levels of promoter methylation in MDGA2-silenced cells and no/low methylation in MDGA2-expressing samples. (C) MDGA2 mRNA expression was restored after treatment with the demethylation reagent 5-Aza. Decreased methylation was revealed by BGS after 5-Aza treatment.
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GUTJNL2015309276F1: MDGA2 is inactivated by promoter methylation in gastric cancer cell lines. (A) MDGA2 was silenced or downregulated in 10 out of 11 gastric cancer cell lines but readily expression in normal gastric tissue. The methylation status of MDGA2 was determined by methylation-specific PCR (MSP). M, methylated; U, unmethylated. (B) A typical CpG island is present at the promoter region of MDGA2. Each vertical bar represents a single CpG site. The transcription start site (TSS) is indicated by a curved arrow. A region for bisulfite genomic sequencing (BGS) and combined bisulfite restriction analysis (COBRA) is denoted. BGS analysis confirmed high levels of promoter methylation in MDGA2-silenced cells and no/low methylation in MDGA2-expressing samples. (C) MDGA2 mRNA expression was restored after treatment with the demethylation reagent 5-Aza. Decreased methylation was revealed by BGS after 5-Aza treatment.

Mentions: We first determined the expression level of MDGA2 by reverse transcription PCR (RT-PCR). MDGA2 was silenced in 10 of the 11 gastric cancer cell lines (90.9%) but was readily expressed in normal human stomach mucosa. Using methylation-specific PCR, almost full methylation was detected in all the 10 silenced gastric cancer cell lines (AGS, BGC823, MGC803, MKN28, MKN45, NCI-N87, SNU1, SNU16, SNU638 and SNU719), whereas methylation was not detected in MKN1 which expressed MDGA2 or normal stomach mucosa (figure 1A). The promoter methylation status of MDGA2 was further evaluated by bisulfite genomic sequencing (BGS). As shown in figure 1B, CpG sites of MDGA2 promoter region were densely methylated in the 10 silenced cell lines (81.5–99.1%), but only 17.8% methylated in MKN1 cells and no methylation was found in normal stomach mucosa. To further validate this, four randomly selected MDGA2-silenced cells (AGS, BGC823, N87 and SNU719) were treated with demethylation agent 5-Aza. MDGA2 expression was subsequently restored in all treated cells (figure 1C). In concordance with the restored MDGA2 expression, the promoter methylation level of MDGA2 was significantly reduced in these cells by 5-Aza treatment (from 94.2±4.7% to 36.0±3.2%; p=0.0002, paired t test). These results show that transcriptional silence of MDGA2 is mediated by promoter hypermethylation in gastric cancer cells.


MDGA2 is a novel tumour suppressor cooperating with DMAP1 in gastric cancer and is associated with disease outcome
MDGA2 is inactivated by promoter methylation in gastric cancer cell lines. (A) MDGA2 was silenced or downregulated in 10 out of 11 gastric cancer cell lines but readily expression in normal gastric tissue. The methylation status of MDGA2 was determined by methylation-specific PCR (MSP). M, methylated; U, unmethylated. (B) A typical CpG island is present at the promoter region of MDGA2. Each vertical bar represents a single CpG site. The transcription start site (TSS) is indicated by a curved arrow. A region for bisulfite genomic sequencing (BGS) and combined bisulfite restriction analysis (COBRA) is denoted. BGS analysis confirmed high levels of promoter methylation in MDGA2-silenced cells and no/low methylation in MDGA2-expressing samples. (C) MDGA2 mRNA expression was restored after treatment with the demethylation reagent 5-Aza. Decreased methylation was revealed by BGS after 5-Aza treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036270&req=5

GUTJNL2015309276F1: MDGA2 is inactivated by promoter methylation in gastric cancer cell lines. (A) MDGA2 was silenced or downregulated in 10 out of 11 gastric cancer cell lines but readily expression in normal gastric tissue. The methylation status of MDGA2 was determined by methylation-specific PCR (MSP). M, methylated; U, unmethylated. (B) A typical CpG island is present at the promoter region of MDGA2. Each vertical bar represents a single CpG site. The transcription start site (TSS) is indicated by a curved arrow. A region for bisulfite genomic sequencing (BGS) and combined bisulfite restriction analysis (COBRA) is denoted. BGS analysis confirmed high levels of promoter methylation in MDGA2-silenced cells and no/low methylation in MDGA2-expressing samples. (C) MDGA2 mRNA expression was restored after treatment with the demethylation reagent 5-Aza. Decreased methylation was revealed by BGS after 5-Aza treatment.
Mentions: We first determined the expression level of MDGA2 by reverse transcription PCR (RT-PCR). MDGA2 was silenced in 10 of the 11 gastric cancer cell lines (90.9%) but was readily expressed in normal human stomach mucosa. Using methylation-specific PCR, almost full methylation was detected in all the 10 silenced gastric cancer cell lines (AGS, BGC823, MGC803, MKN28, MKN45, NCI-N87, SNU1, SNU16, SNU638 and SNU719), whereas methylation was not detected in MKN1 which expressed MDGA2 or normal stomach mucosa (figure 1A). The promoter methylation status of MDGA2 was further evaluated by bisulfite genomic sequencing (BGS). As shown in figure 1B, CpG sites of MDGA2 promoter region were densely methylated in the 10 silenced cell lines (81.5–99.1%), but only 17.8% methylated in MKN1 cells and no methylation was found in normal stomach mucosa. To further validate this, four randomly selected MDGA2-silenced cells (AGS, BGC823, N87 and SNU719) were treated with demethylation agent 5-Aza. MDGA2 expression was subsequently restored in all treated cells (figure 1C). In concordance with the restored MDGA2 expression, the promoter methylation level of MDGA2 was significantly reduced in these cells by 5-Aza treatment (from 94.2±4.7% to 36.0±3.2%; p=0.0002, paired t test). These results show that transcriptional silence of MDGA2 is mediated by promoter hypermethylation in gastric cancer cells.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Using the promoter methylation assay, we have shown that MDGA2 (MAM domain containing glycosylphosphatidylinositol anchor 2) is preferentially methylated in gastric cancer. We analysed its biological effects and prognostic significance in gastric cancer.

Methods: MDGA2 methylation status was evaluated by combined bisulfite restriction analysis and bisulfite genomic sequencing. The effects of MDGA2 re-expression or knockdown on cell proliferation, apoptosis and the cell cycle were determined. MDGA2 interacting protein was identified by mass spectrometry and MDGA2-related cancer pathways by reporter activity and PCR array analyses. The clinical impact of MDGA2 was assessed in 218 patients with gastric cancer.

Results: MDGA2 was commonly silenced in gastric cancer cells (10/11) and primary gastric cancers due to promoter hypermethylation. MDGA2 significantly inhibited cell proliferation by causing G1–S cell cycle arrest and inducing cell apoptosis in vitro, and suppressed xenograft tumour growth in both subcutaneous and orthotopic xenograft mouse models (both p<0.001). The anti-tumorigenic effect of MDGA2 was mediated through direct stabilising of DNA methyltransferase 1 associated protein 1 (DMAP1), which played a tumour suppressive role in gastric cancer. This interaction activated their downstream key elements of p53/p21 signalling cascades. Moreover, promoter methylation of MDGA2 was detected in 62.4% (136/218) of gastric cancers. Multivariate analysis showed that patients with MDGA2 hypermethylation had a significantly decreased survival (p=0.005). Kaplan–Meier survival curves showed that MDGA2 hypermethylation was significantly associated with shortened survival in patients with early gastric cancer.

Conclusions: MDGA2 is a critical tumour suppressor in gastric carcinogenesis; its hypermethylation is an independent prognostic factor in patients with gastric cancer.

No MeSH data available.


Related in: MedlinePlus