Net1 and Myeov: computationally identified mediators of gastric 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.

NET1-mediated RhoA activation facilitates lysophosphatidic acid-induced cell migration and invasion in gastric cancer

“…Cell culture and cell treatments AGS gastric cancer cell lines were cultured in Hams F12 medium supplemented with 10% fetal bovine serum and grown under standard conditions as previously described (Leyden et al, 2006). Throughout this study, cells were treated with the following: 20 mM LPA for 4 h; 4 mg ml À1 C3 exoenzyme for 4 h or 5 mM cytochalasin D (CyD).…”

“…Neuroepithelial cell transforming gene 1 mRNA was silenced as previously described (Leyden et al, 2006). Briefly, siRNA duplexes were designed and synthesised for transiently silencing NET1, and a chemically synthesised non-silencing siRNA duplex that had no known homology with any mammalian gene was used to control nonspecific silencing events (Qiagen Inc., Valencia, CA, USA).…”

“…TRIzolt (Sigma-Aldrich, Dublin, Ireland) was used to extract RNA as previously described (Leyden et al, 2006). Following reverse transcription, as previously described (Leyden et al, 2006) real-time PCR was performed using a QuantiTectt SYBR Green PCR Kit (Qiagen) following the manufacturers' instructions.…”

“…Following reverse transcription, as previously described (Leyden et al, 2006) real-time PCR was performed using a QuantiTectt SYBR Green PCR Kit (Qiagen) following the manufacturers' instructions. Briefly, 2 ml of cDNA template was mixed with 12.5 ml SYBR Green master mix containing Taq and dNTPs (Qiagen), 8.5 ml DNase-free water and 1 ml each of forward and reverse primers.…”

“…We have recently identified enhanced NET1 expression in GA in comparison with adjacent normal tissue and we have furthermore shown NET1 to play a role in tumour cell invasion (Leyden et al, 2006). Neuroepithelial cell transforming gene 1 is a member of the guanine nucleotide exchange factor (GEF) family, a group of proteins that are known to activate and thereby regulate Rho family members (Symons and Rusk, 2003;Rossman et al, 2005).…”