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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

In vitro gain- and loss-of-function assays on MDGA2. (A) Ectopic expression of MDGA2 in AGS and BGC823 cells at mRNA and protein levels was confirmed by reverse transcription PCR (RT-PCR) and western blot analysis. (B) MDGA2 significantly inhibited cell viability and colony formation ability. (C) MDGA2 caused cell cycle arrest at G1–S transition, as indicated by flow cytometry. Reduced cell proliferation by MDGA2 was further shown by Ki-67 staining (magnification ×400) and altered cell cycle-related protein expression by western blot analysis. (D) Cell apoptosis by flow cytometry analysis after annexin V-APC and 7-aminoactinomycin (7-AAD) double staining. Q2 shows the late apoptotic cells and Q4 shows the early apoptotic cells. Cell apoptosis was confirmed by terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) staining (magnification ×400) and upregulation of apoptosis-related proteins in MDGA2-expressing cells was confirmed by western blot analysis. (E) Growth inhibitory effect of cisplatin on MDGA2-overexpressing and control vector-transfected AGS and BGC823 cells. Cell viability was measured by MTT assay after 48 h treatment with cisplatin. Data are mean±SD from three independent experiments. (F) Knockdown of MDGA2 in MKN1 cells by short interference RNA (siRNA) transfection was confirmed by RT-PCR, qRT-PCR and western blot analysis. (G) Knockdown of MDGA2 significantly increased cell viability of MKN1 cells, promoted colony formation and (H) promoted cell cycle progression, but reduced cell apoptosis as indicated by flow cytometry. *p<0.05, **p<0.01, ***p<0.001.
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GUTJNL2015309276F3: In vitro gain- and loss-of-function assays on MDGA2. (A) Ectopic expression of MDGA2 in AGS and BGC823 cells at mRNA and protein levels was confirmed by reverse transcription PCR (RT-PCR) and western blot analysis. (B) MDGA2 significantly inhibited cell viability and colony formation ability. (C) MDGA2 caused cell cycle arrest at G1–S transition, as indicated by flow cytometry. Reduced cell proliferation by MDGA2 was further shown by Ki-67 staining (magnification ×400) and altered cell cycle-related protein expression by western blot analysis. (D) Cell apoptosis by flow cytometry analysis after annexin V-APC and 7-aminoactinomycin (7-AAD) double staining. Q2 shows the late apoptotic cells and Q4 shows the early apoptotic cells. Cell apoptosis was confirmed by terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) staining (magnification ×400) and upregulation of apoptosis-related proteins in MDGA2-expressing cells was confirmed by western blot analysis. (E) Growth inhibitory effect of cisplatin on MDGA2-overexpressing and control vector-transfected AGS and BGC823 cells. Cell viability was measured by MTT assay after 48 h treatment with cisplatin. Data are mean±SD from three independent experiments. (F) Knockdown of MDGA2 in MKN1 cells by short interference RNA (siRNA) transfection was confirmed by RT-PCR, qRT-PCR and western blot analysis. (G) Knockdown of MDGA2 significantly increased cell viability of MKN1 cells, promoted colony formation and (H) promoted cell cycle progression, but reduced cell apoptosis as indicated by flow cytometry. *p<0.05, **p<0.01, ***p<0.001.

Mentions: To elucidate the tumour suppressive function of MDGA2 in gastric cancer, MDGA2 expression vector was stably transfected into the MDGA2-silenced AGS and BGC823 cells, with empty vector transfection as control. Re-expression of MDGA2 mRNA and protein in these cells was evidenced by RT-PCR and western blot analysis (figure 3A). Ectopic expression of MDGA2 significantly inhibited cell growth, as evidenced by cell viability and colony formation assays in AGS and BGC823 (figure 3B).


MDGA2 is a novel tumour suppressor cooperating with DMAP1 in gastric cancer and is associated with disease outcome
In vitro gain- and loss-of-function assays on MDGA2. (A) Ectopic expression of MDGA2 in AGS and BGC823 cells at mRNA and protein levels was confirmed by reverse transcription PCR (RT-PCR) and western blot analysis. (B) MDGA2 significantly inhibited cell viability and colony formation ability. (C) MDGA2 caused cell cycle arrest at G1–S transition, as indicated by flow cytometry. Reduced cell proliferation by MDGA2 was further shown by Ki-67 staining (magnification ×400) and altered cell cycle-related protein expression by western blot analysis. (D) Cell apoptosis by flow cytometry analysis after annexin V-APC and 7-aminoactinomycin (7-AAD) double staining. Q2 shows the late apoptotic cells and Q4 shows the early apoptotic cells. Cell apoptosis was confirmed by terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) staining (magnification ×400) and upregulation of apoptosis-related proteins in MDGA2-expressing cells was confirmed by western blot analysis. (E) Growth inhibitory effect of cisplatin on MDGA2-overexpressing and control vector-transfected AGS and BGC823 cells. Cell viability was measured by MTT assay after 48 h treatment with cisplatin. Data are mean±SD from three independent experiments. (F) Knockdown of MDGA2 in MKN1 cells by short interference RNA (siRNA) transfection was confirmed by RT-PCR, qRT-PCR and western blot analysis. (G) Knockdown of MDGA2 significantly increased cell viability of MKN1 cells, promoted colony formation and (H) promoted cell cycle progression, but reduced cell apoptosis as indicated by flow cytometry. *p<0.05, **p<0.01, ***p<0.001.
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GUTJNL2015309276F3: In vitro gain- and loss-of-function assays on MDGA2. (A) Ectopic expression of MDGA2 in AGS and BGC823 cells at mRNA and protein levels was confirmed by reverse transcription PCR (RT-PCR) and western blot analysis. (B) MDGA2 significantly inhibited cell viability and colony formation ability. (C) MDGA2 caused cell cycle arrest at G1–S transition, as indicated by flow cytometry. Reduced cell proliferation by MDGA2 was further shown by Ki-67 staining (magnification ×400) and altered cell cycle-related protein expression by western blot analysis. (D) Cell apoptosis by flow cytometry analysis after annexin V-APC and 7-aminoactinomycin (7-AAD) double staining. Q2 shows the late apoptotic cells and Q4 shows the early apoptotic cells. Cell apoptosis was confirmed by terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) staining (magnification ×400) and upregulation of apoptosis-related proteins in MDGA2-expressing cells was confirmed by western blot analysis. (E) Growth inhibitory effect of cisplatin on MDGA2-overexpressing and control vector-transfected AGS and BGC823 cells. Cell viability was measured by MTT assay after 48 h treatment with cisplatin. Data are mean±SD from three independent experiments. (F) Knockdown of MDGA2 in MKN1 cells by short interference RNA (siRNA) transfection was confirmed by RT-PCR, qRT-PCR and western blot analysis. (G) Knockdown of MDGA2 significantly increased cell viability of MKN1 cells, promoted colony formation and (H) promoted cell cycle progression, but reduced cell apoptosis as indicated by flow cytometry. *p<0.05, **p<0.01, ***p<0.001.
Mentions: To elucidate the tumour suppressive function of MDGA2 in gastric cancer, MDGA2 expression vector was stably transfected into the MDGA2-silenced AGS and BGC823 cells, with empty vector transfection as control. Re-expression of MDGA2 mRNA and protein in these cells was evidenced by RT-PCR and western blot analysis (figure 3A). Ectopic expression of MDGA2 significantly inhibited cell growth, as evidenced by cell viability and colony formation assays in AGS and BGC823 (figure 3B).

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&ndash;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&lt;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&ndash;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