Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
. 2006 Apr 24;94(8):1204-12.
doi: 10.1038/sj.bjc.6603054.

Net1 and Myeov: computationally identified mediators of gastric cancer

J Leyden  1 , D Murray, A Moss, M Arumuguma, E Doyle, G McEntee, C O'Keane, P Doran, P MacMathuna
Affiliations
Comparative Study

Net1 and Myeov: computationally identified mediators of gastric cancer

J Leyden et al. Br J Cancer. .

Abstract

Gastric adenocarcinoma (GA) is a significant cause of mortality worldwide. The molecular mechanisms of GA remain poorly characterised. Our aim was to characterise the functional activity of the computationally identified genes, NET 1 and MYEOV in GA. Digital Differential Display was used to identify genes altered expression in GA-derived EST libraries. mRNA levels of a subset of genes were quantitated by qPCR in a panel of cell lines and tumour tissue. The effect of pro- and anti-inflammatory stimuli on gene expression was investigated. Cell proliferation and invasion were measured using in an in-vitro GA model following inhibition of expression using siRNA. In all, 23 genes not previously reported in association with GA were identified. Two genes, Net1 and Myeov, were selected for further analysis and increased expression was detected in GA tissue compared to paired normal tissue using quantitative PCR. siRNA-mediated downregulation of Net1 and Myeov resulted in decreased proliferation and invasion of gastric cancer cells in vitro. These functional studies highlight a putative role for NET1 and Myeov in the development and progression of gastric cancer. These genes may provide important targets for intervention in GA, evidenced by their role in promoting invasion and proliferation, key phenotypic hallmarks of cancer cells.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Net1 expression in paired gastric normal and tumour tissue. Real-time PCR was used to investigate the levels of Net1 expression in matched gastric cancer (C) and normal (N) tissue. Each tissue specimen was analysed in triplicate for mRNA levels. β-actin expression was used to normalise the data. *(P<0.05). Average Net1 expression in cancer (AC) and normal (AN) tissue is displayed.
Figure 2
Figure 2
Net1 expression in normal epithelial and gastric cancer cell lines. Real-time PCR was used to determine the relative expression levels of Net1 mRNA in normal alveolar epithelial cells (AEC) and in three gastric cancer cell lines (AGS, 23132/87 (231) and KatoIII (Kato). Each sample was analysed in triplicate for mRNA levels. β-actin expression was used to normalise the data.
Figure 3
Figure 3
Myeov expression in paired gastric normal and tumour tissue. Real-time PCR was used to investigate the levels of Myeov expression in paired gastric cancer (C) and normal (N) tissue. Each tissue specimen was analysed in triplicate for mRNA levels. β-actin expression levels were used to normalise the data. β-actin expression was used to normalise all data. *(P<0.05). Average Myeov expression in cancer (AC) and normal (AN) tissue is displayed.
Figure 4
Figure 4
Myeov expression in normal epithelial and gastric cancer cell lines. Real-time PCR was used to determine the relative expression levels of Myeov mRNA in normal alveolar epithelial cells (AEC) and in three gastric cancer cell lines (AGS, 23132/87 (231) and KatoIII (Kato). Each sample was analysed in triplicate and β-actin expression was used to normalise the data.
Figure 5
Figure 5
The effect of pro- and anti-inflammatory stimuli on Net1 and Myeov expression in gastric cancer. (A) The effect of 10 ng ml−1 interleukin-1β (IL-1B), TNF-alpha (TNFa) and Dexamethasone (DEX) on Net1 and Myeov expression in AGS gastric cancer cells was monitored using real-time PCR. Cells were treated for 4 h. Enhanced TNFa expression was used as a positive response to TNFa and IL1B treatment (Gallagher et al, 2003). Enhanced MKP1 expression was used a positive response to DEX treatment (Lasa et al, 2002). Apart from the positive controls, the only trteatment to have a significant effect was TNFα, which resulted in a threefold increase in Net1 mRNA expression *(P<0.05). Enhanced TNFa-induced Net1 expression resulted in a dose-dependent (B) and time-dependent (C) manner *(P<0.05).
Figure 6
Figure 6
The effect of Net1 knockdown on gastric adenocarcinoma cell proliferation and invasion. (A) Real-time PCR was used to confirm the significant siRNA-mediated reduction in Net1 expression. Using two siRNA duplexes; Net1(1) and Net1(2), Net 1 expression was reduced by 49 and 41%, respectively, in comparison with control cells. Using a combination of both siRNA duplexes; Net1(1+2) resulted in 55% decreased Net1 expression (P<0.05). (B) The proliferation of cells treated with both duplexes was compared with control cells using an MTS assay. siRNA mediated Net1 downregulation resulted in 47 and 41% decrease in cell proliferation. Using both Net1 siRNA duplexes in combination resulted in 58% decreased cell proliferation (P<0.05). (C) Downregulation of Net1 expression using both siRNA duplexes resulted in 100 and 96% reduction in cell invasion in comparison with control cells. A combination of both siRNA molecules resulted in 96% AGS cell invasion *(P<0.05).
Figure 7
Figure 7
The effect of siRNA-mediated Myeov knockdown on AGS cell proliferation and invasion. (A) Confirmation of Myeov downregulation using real-time PCR. Myeov downregulation, using two separate siRNA duplexes, Myeov(1) and Myeov(2), resulted in 100 and 30% decreased expression when compared with control cells. A combination of both Myeov siRNA molecules resulted in 75% decreased Myeov expression (P<0.05). (B) Using siRNA duplexes, cell proliferation was reduced 68 and 36%, respectively, when compared with control cells. Using both duplexes in combination resulted in 73% decrease in gastric cancer cell proliferation (P<0.05). (C) Similarly, cell invasion was reduced by 85 and 90% and by 99% using a combination of both siRNA duplexes in comparison with control cells *(P<0.05).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Advani AS, Pendergast AM (2002) Bcr–Abl variants: biological and clinical aspects. Leuk Res 26: 713–720 - PubMed
    1. Alberts AS, Treisman R (1998) Activation of RhoA and SAPK/JNK signalling pathways by the RhoA-specific exchange factor mNET1. EMBO J 17: 4075–4085 - PMC - PubMed
    1. Barranco SC, Townsend Jr CM, Casartelli C, Macik BG, Burger NL, Boerwinkle WR, Gourley WK (1983) Establishment and characterization of an in vitro model system for human adenocarcinoma of the stomach. Cancer Res 43: 1703–1709 - PubMed
    1. Benbow U, Brinckerhoff CE (1997) The AP-1 site and MMP gene regulation: what is all the fuss about? Matrix Biol 15: 519–526 - PubMed
    1. Bretscher A, Edwards K, Fehon RG (2002) ERM proteins and merlin: integrators at the cell cortex. Nat Rev Mol Cell Biol 3: 586–599 - PubMed

Publication types

MeSH terms

-