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Identification of retinoic acid-regulated nuclear matrix-associated protein as a novel regulator of gastric cancer.

Li J, Ng EK, Ng YP, Wong CY, Yu J, Jin H, Cheng VY, Go MY, Cheung PK, Ebert MP, Tong J, To KF, Chan FK, Sung JJ, Ip NY, Leung WK - Br. J. Cancer (2009)

Bottom Line: Ramp was readily expressed in all seven gastric cancer cell lines and was significantly increased in human gastric cancer tissues when compared with their adjacent non-cancerous tissues (P<0.001).In keeping with this, expression of RAMP protein was higher in gastric cancer tissues compared with their adjacent non-cancerous tissues, whereas moderate protein expression were noted in intestinal metaplasia.Knockdown of RAMP in gastric cancer cells significantly reduced cell proliferation (P<0.01) and soft agar colony formation (P<0.001), but induced apoptosis and G(2)/M arrest.

View Article: PubMed Central - PubMed

Affiliation: Institute of Digestive Disease, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong, China.

ABSTRACT

Background: Retinoic acid-regulated nuclear matrix-associated protein (RAMP) is a WD40 repeat-containing protein that is involved in various biological functions, but little is known about its role in human cancer. This study aims to delineate the oncogenic role of RAMP in gastric carcinogenesis.

Methods: RAMP expression was examined by real-time quantitative RT-PCR, immunohistochemistry and western blotting. Inhibition of RAMP expression was performed by siRNA-mediated knockdown. The functional effects of RAMP on cell kinetics were measured by cell viability assay, colony formation assay and flow cytometry. Cell lines stably expressing RAMP were established to investigate the oncogenic effects of RAMP in vitro.

Results: Ramp was readily expressed in all seven gastric cancer cell lines and was significantly increased in human gastric cancer tissues when compared with their adjacent non-cancerous tissues (P<0.001). In keeping with this, expression of RAMP protein was higher in gastric cancer tissues compared with their adjacent non-cancerous tissues, whereas moderate protein expression were noted in intestinal metaplasia. Knockdown of RAMP in gastric cancer cells significantly reduced cell proliferation (P<0.01) and soft agar colony formation (P<0.001), but induced apoptosis and G(2)/M arrest. In additional, knockdown RAMP induced cell apoptosis is dependent on functional accumulation of p53 and p21 and induction of cleaved caspases-9, caspases-3 and PARP. Strikingly, overexpression of RAMP promoted anchorage-independent cell growth in soft agar.

Conclusion: Our findings demonstrate that RAMP plays an oncogenic role in gastric carcinogenesis. Inhibition of RAMP may be a promising approach for gastric cancer therapy.

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Related in: MedlinePlus

Overexpression of RAMP promoted cell colony formation in soft agar assay. (A) MKN28 and (B) Rat2 were stably transfected with RAMP-expressing or empty vectors. Expression of RAMP in transfected cells was confirmed by western blotting. Assays were performed in triplicate for three times. Quantitative analyses of colony numbers are shown as values of mean±s.d.; *P<0.05.
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fig5: Overexpression of RAMP promoted cell colony formation in soft agar assay. (A) MKN28 and (B) Rat2 were stably transfected with RAMP-expressing or empty vectors. Expression of RAMP in transfected cells was confirmed by western blotting. Assays were performed in triplicate for three times. Quantitative analyses of colony numbers are shown as values of mean±s.d.; *P<0.05.

Mentions: To further investigate the oncogenic potential of RAMP in gastric cancer, we examined the effect of RAMP overexpression on growth characteristics of gastric tumour cells using soft agar colony formation assays. RAMP expression vector was stably transfected into MKN28 cells, which exhibits the lowest RAMP expression among seven gastric cancer cell lines, and Rat2 cells, an immortalised normal rat fibroblast cell line. Overexpression of RAMP in the transfected cells was confirmed by western blotting (Figure 5). Our results demonstrated that the colony formation efficiencies from the RAMP-transfected MKN28 and Rat2 cells were significantly higher (P<0.05) and larger in size than those of empty vector transfected cells (Figure 5).


Identification of retinoic acid-regulated nuclear matrix-associated protein as a novel regulator of gastric cancer.

Li J, Ng EK, Ng YP, Wong CY, Yu J, Jin H, Cheng VY, Go MY, Cheung PK, Ebert MP, Tong J, To KF, Chan FK, Sung JJ, Ip NY, Leung WK - Br. J. Cancer (2009)

Overexpression of RAMP promoted cell colony formation in soft agar assay. (A) MKN28 and (B) Rat2 were stably transfected with RAMP-expressing or empty vectors. Expression of RAMP in transfected cells was confirmed by western blotting. Assays were performed in triplicate for three times. Quantitative analyses of colony numbers are shown as values of mean±s.d.; *P<0.05.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2736823&req=5

fig5: Overexpression of RAMP promoted cell colony formation in soft agar assay. (A) MKN28 and (B) Rat2 were stably transfected with RAMP-expressing or empty vectors. Expression of RAMP in transfected cells was confirmed by western blotting. Assays were performed in triplicate for three times. Quantitative analyses of colony numbers are shown as values of mean±s.d.; *P<0.05.
Mentions: To further investigate the oncogenic potential of RAMP in gastric cancer, we examined the effect of RAMP overexpression on growth characteristics of gastric tumour cells using soft agar colony formation assays. RAMP expression vector was stably transfected into MKN28 cells, which exhibits the lowest RAMP expression among seven gastric cancer cell lines, and Rat2 cells, an immortalised normal rat fibroblast cell line. Overexpression of RAMP in the transfected cells was confirmed by western blotting (Figure 5). Our results demonstrated that the colony formation efficiencies from the RAMP-transfected MKN28 and Rat2 cells were significantly higher (P<0.05) and larger in size than those of empty vector transfected cells (Figure 5).

Bottom Line: Ramp was readily expressed in all seven gastric cancer cell lines and was significantly increased in human gastric cancer tissues when compared with their adjacent non-cancerous tissues (P<0.001).In keeping with this, expression of RAMP protein was higher in gastric cancer tissues compared with their adjacent non-cancerous tissues, whereas moderate protein expression were noted in intestinal metaplasia.Knockdown of RAMP in gastric cancer cells significantly reduced cell proliferation (P<0.01) and soft agar colony formation (P<0.001), but induced apoptosis and G(2)/M arrest.

View Article: PubMed Central - PubMed

Affiliation: Institute of Digestive Disease, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong, China.

ABSTRACT

Background: Retinoic acid-regulated nuclear matrix-associated protein (RAMP) is a WD40 repeat-containing protein that is involved in various biological functions, but little is known about its role in human cancer. This study aims to delineate the oncogenic role of RAMP in gastric carcinogenesis.

Methods: RAMP expression was examined by real-time quantitative RT-PCR, immunohistochemistry and western blotting. Inhibition of RAMP expression was performed by siRNA-mediated knockdown. The functional effects of RAMP on cell kinetics were measured by cell viability assay, colony formation assay and flow cytometry. Cell lines stably expressing RAMP were established to investigate the oncogenic effects of RAMP in vitro.

Results: Ramp was readily expressed in all seven gastric cancer cell lines and was significantly increased in human gastric cancer tissues when compared with their adjacent non-cancerous tissues (P<0.001). In keeping with this, expression of RAMP protein was higher in gastric cancer tissues compared with their adjacent non-cancerous tissues, whereas moderate protein expression were noted in intestinal metaplasia. Knockdown of RAMP in gastric cancer cells significantly reduced cell proliferation (P<0.01) and soft agar colony formation (P<0.001), but induced apoptosis and G(2)/M arrest. In additional, knockdown RAMP induced cell apoptosis is dependent on functional accumulation of p53 and p21 and induction of cleaved caspases-9, caspases-3 and PARP. Strikingly, overexpression of RAMP promoted anchorage-independent cell growth in soft agar.

Conclusion: Our findings demonstrate that RAMP plays an oncogenic role in gastric carcinogenesis. Inhibition of RAMP may be a promising approach for gastric cancer therapy.

Show MeSH
Related in: MedlinePlus