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A functional and transcriptomic analysis of NET1 bioactivity in gastric cancer

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 sta...

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Autores principales: Bennett, Gayle, Sadlier, Denise, Doran, Peter P, MacMathuna, Padraic, Murray, David W
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3041777/
https://www.ncbi.nlm.nih.gov/pubmed/21284875
http://dx.doi.org/10.1186/1471-2407-11-50
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author Bennett, Gayle
Sadlier, Denise
Doran, Peter P
MacMathuna, Padraic
Murray, David W
author_facet Bennett, Gayle
Sadlier, Denise
Doran, Peter P
MacMathuna, Padraic
Murray, David W
author_sort Bennett, Gayle
collection PubMed
description 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.
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spelling pubmed-30417772011-02-19 A functional and transcriptomic analysis of NET1 bioactivity in gastric cancer Bennett, Gayle Sadlier, Denise Doran, Peter P MacMathuna, Padraic Murray, David W BMC Cancer Research Article 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. BioMed Central 2011-02-01 /pmc/articles/PMC3041777/ /pubmed/21284875 http://dx.doi.org/10.1186/1471-2407-11-50 Text en Copyright ©2011 Bennett et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Bennett, Gayle
Sadlier, Denise
Doran, Peter P
MacMathuna, Padraic
Murray, David W
A functional and transcriptomic analysis of NET1 bioactivity in gastric cancer
title A functional and transcriptomic analysis of NET1 bioactivity in gastric cancer
title_full A functional and transcriptomic analysis of NET1 bioactivity in gastric cancer
title_fullStr A functional and transcriptomic analysis of NET1 bioactivity in gastric cancer
title_full_unstemmed A functional and transcriptomic analysis of NET1 bioactivity in gastric cancer
title_short A functional and transcriptomic analysis of NET1 bioactivity in gastric cancer
title_sort functional and transcriptomic analysis of net1 bioactivity in gastric cancer
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3041777/
https://www.ncbi.nlm.nih.gov/pubmed/21284875
http://dx.doi.org/10.1186/1471-2407-11-50
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