title: A functional and transcriptomic analysis of NET1 bioactivity in gastric cancer.
abstract: BACKGROUND
  NET1, a RhoA guanine exchange factor, is up-regulated in gastric cancer (GC) tissue and drives the invasive phenotype of this disease. In this study, we aimed to determine the role of NET1 in GC by monitoring the proliferation, motility and invasion of GC cells in which NET1 has been stably knocked down. Additionally, we aimed to determine NET1-dependent transcriptomic events that occur in GC.
  
  
  METHODS
  An in vitro model of stable knockdown of NET1 was achieved in AGS human gastric adenocarcinoma cells via lentiviral mediated transduction of short-hairpin (sh) RNA targeting NET1. Knockdown was assessed using quantitative PCR. Cell proliferation was assessed using an MTS assay and cell migration was assessed using a wound healing scratch assay. Cell invasion was assessed using a transwell matrigel invasion assay. Gene expression profiles were examined using affymetrix oligonucleotide U133A expression arrays. A student's t test was used to determine changes of statistical significance.
  
  
  RESULTS
  GC cells were transduced with NET1 shRNA resulting in a 97% reduction in NET1 mRNA (p < 0.0001). NET1 knockdown significantly reduced the invasion and migration of GC cells by 94% (p < 0.05) and 24% (p < 0.001) respectively, while cell proliferation was not significantly altered following NET1 knockdown. Microarray analysis was performed on non-target and knockdown cell lines, treated with and without 10 μM lysophosphatidic acid (LPA) allowing us to identify NET1-dependent, LPA-dependent and NET1-mediated LPA-induced gene transcription. Differential gene expression was confirmed by quantitative PCR. Shortlisted NET1-dependent genes included STAT1, TSPAN1, TGFBi and CCL5 all of which were downregulatd upon NET1 downregulation. Shortlisted LPA-dependent genes included EGFR and PPARD where EGFR was upregulated and PPARD was downregulated upon LPA stimulation. Shortlisted NET1 and LPA dependent genes included IGFR1 and PIP5K3. These LPA induced genes were downregulated in NET1 knockdown cells.
  
  
  CONCLUSIONS
  NET1 plays an important role in GC cell migration and invasion, key aspects of GC progression. Furthermore, the gene expression profile further elucidates the molecular mechanisms underpinning NET1-mediated aggressive GC cell behaviour.
authors.affiliation: UCD Clinical Research Centre, UCD School of Medicine and Medical Sciences, Misericordiae University Hospital, Dublin 7, Ireland.
authors.forename: Gayle
authors.initials: G
authors.lastname: Bennett
authors.forename: Denise
authors.initials: D
authors.lastname: Sadlier
authors.forename: Peter P
authors.initials: PP
authors.lastname: Doran
authors.forename: Padraic
authors.initials: P
authors.lastname: Macmathuna
authors.forename: David W
authors.initials: DW
authors.lastname: Murray
mesh_terms: D000230:Adenocarcinoma / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D048708:Cell Growth Processes / Q000502:physiology; D002465:Cell Movement / Q000502:physiology; D020869:Gene Expression Profiling; D055785:Gene Knockdown Techniques; D006801:Humans; D009361:Neoplasm Invasiveness; D015513:Oncogene Proteins / Q000096:biosynthesis / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013274:Stomach Neoplasms / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D014407:Tumor Cells, Cultured
chemical_list: C099763:NET1 protein, human; D015513:Oncogene Proteins; D034741:RNA, Small Interfering