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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 vivo subcutaneous and orthotopic xenograft models confirmed the suppressive effect of MDGA2 on tumorigenicity. (A) Subcutaneous tumour growth curve of MDGA2-expressing BGC823 cells in nude mice was compared with control vector (pcDNA3.1) transfected cells. The data are mean±SD (n=5/group) of three separate experiments. (B) A representative image of tumour growth in nude mice subcutaneously inoculated with MDGA2- or control vector-transfected cells. Tumour weight was compared at the end of the experiment. MDGA2 expression in subcutaneous xenografts was confirmed by immunohistochemistry (IHC). (C) Cell proliferative activity was evaluated by Ki-67 staining and apoptotic activity by terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) staining in subcutaneous xenografts. (D) Representative images of orthotopic xenograft tumours. Both volume and weight of the orthotopic xenograft tumours were significantly smaller in the MDGA2 group than in the control group. (E) Confirmation of MDGA2 expression in orthotopic xenograft tumours by IHC. (F) Ki-67 staining in orthotopic xenografts and TUNEL staining in orthotopic xenografts. *p<0.05, **p<0.001, ***p<0.0001.
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GUTJNL2015309276F4: In vivo subcutaneous and orthotopic xenograft models confirmed the suppressive effect of MDGA2 on tumorigenicity. (A) Subcutaneous tumour growth curve of MDGA2-expressing BGC823 cells in nude mice was compared with control vector (pcDNA3.1) transfected cells. The data are mean±SD (n=5/group) of three separate experiments. (B) A representative image of tumour growth in nude mice subcutaneously inoculated with MDGA2- or control vector-transfected cells. Tumour weight was compared at the end of the experiment. MDGA2 expression in subcutaneous xenografts was confirmed by immunohistochemistry (IHC). (C) Cell proliferative activity was evaluated by Ki-67 staining and apoptotic activity by terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) staining in subcutaneous xenografts. (D) Representative images of orthotopic xenograft tumours. Both volume and weight of the orthotopic xenograft tumours were significantly smaller in the MDGA2 group than in the control group. (E) Confirmation of MDGA2 expression in orthotopic xenograft tumours by IHC. (F) Ki-67 staining in orthotopic xenografts and TUNEL staining in orthotopic xenografts. *p<0.05, **p<0.001, ***p<0.0001.

Mentions: To examine the effect of MDGA2 on gastric tumour growth in vivo, we first set up subcutaneous xenograft tumour models by subcutaneously injecting MDGA2-transfected and empty vector-transfected BGC823 cells in nude mice. Subcutaneous tumour growth was then monitored and compared between the two groups. As shown in figure 4A, the growth of tumour volume by MDGA2-transfected cells was significantly suppressed compared with control cells (p<0.0001). The net weight of tumours formed by MDGA2-transfected cells was also significantly reduced compared with controls at termination of the experiment (p<0.01; figure 4B). Protein expression of MDGA2 was detected in xenograft tumours of the MDGA2-transfected group but not in the control group by immunohistochemistry (figure 4B), indicating that the tumour suppressive effect was exerted by MDGA2 expression. In concordance with the in vitro findings, significantly fewer proliferating cells and more apoptotic cells were detected in MDGA2-expressed xenografts, as indicated by Ki-67 and TUNEL assays, respectively (both p<0.001; figure 4C).


MDGA2 is a novel tumour suppressor cooperating with DMAP1 in gastric cancer and is associated with disease outcome
In vivo subcutaneous and orthotopic xenograft models confirmed the suppressive effect of MDGA2 on tumorigenicity. (A) Subcutaneous tumour growth curve of MDGA2-expressing BGC823 cells in nude mice was compared with control vector (pcDNA3.1) transfected cells. The data are mean±SD (n=5/group) of three separate experiments. (B) A representative image of tumour growth in nude mice subcutaneously inoculated with MDGA2- or control vector-transfected cells. Tumour weight was compared at the end of the experiment. MDGA2 expression in subcutaneous xenografts was confirmed by immunohistochemistry (IHC). (C) Cell proliferative activity was evaluated by Ki-67 staining and apoptotic activity by terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) staining in subcutaneous xenografts. (D) Representative images of orthotopic xenograft tumours. Both volume and weight of the orthotopic xenograft tumours were significantly smaller in the MDGA2 group than in the control group. (E) Confirmation of MDGA2 expression in orthotopic xenograft tumours by IHC. (F) Ki-67 staining in orthotopic xenografts and TUNEL staining in orthotopic xenografts. *p<0.05, **p<0.001, ***p<0.0001.
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Related In: Results  -  Collection

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GUTJNL2015309276F4: In vivo subcutaneous and orthotopic xenograft models confirmed the suppressive effect of MDGA2 on tumorigenicity. (A) Subcutaneous tumour growth curve of MDGA2-expressing BGC823 cells in nude mice was compared with control vector (pcDNA3.1) transfected cells. The data are mean±SD (n=5/group) of three separate experiments. (B) A representative image of tumour growth in nude mice subcutaneously inoculated with MDGA2- or control vector-transfected cells. Tumour weight was compared at the end of the experiment. MDGA2 expression in subcutaneous xenografts was confirmed by immunohistochemistry (IHC). (C) Cell proliferative activity was evaluated by Ki-67 staining and apoptotic activity by terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) staining in subcutaneous xenografts. (D) Representative images of orthotopic xenograft tumours. Both volume and weight of the orthotopic xenograft tumours were significantly smaller in the MDGA2 group than in the control group. (E) Confirmation of MDGA2 expression in orthotopic xenograft tumours by IHC. (F) Ki-67 staining in orthotopic xenografts and TUNEL staining in orthotopic xenografts. *p<0.05, **p<0.001, ***p<0.0001.
Mentions: To examine the effect of MDGA2 on gastric tumour growth in vivo, we first set up subcutaneous xenograft tumour models by subcutaneously injecting MDGA2-transfected and empty vector-transfected BGC823 cells in nude mice. Subcutaneous tumour growth was then monitored and compared between the two groups. As shown in figure 4A, the growth of tumour volume by MDGA2-transfected cells was significantly suppressed compared with control cells (p<0.0001). The net weight of tumours formed by MDGA2-transfected cells was also significantly reduced compared with controls at termination of the experiment (p<0.01; figure 4B). Protein expression of MDGA2 was detected in xenograft tumours of the MDGA2-transfected group but not in the control group by immunohistochemistry (figure 4B), indicating that the tumour suppressive effect was exerted by MDGA2 expression. In concordance with the in vitro findings, significantly fewer proliferating cells and more apoptotic cells were detected in MDGA2-expressed xenografts, as indicated by Ki-67 and TUNEL assays, respectively (both p<0.001; figure 4C).

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