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British Journal of Cancer (2007) 96, 464-473.
doi:10.1038/sj.bjc.6603588 www.bjcancer.com
Use of interfering RNA to investigate the role of endogenous gastrin in the survival of gastrointestinal cancer cells
A M Grabowska1, J Hughes1 and S A Watson1
1Academic Unit of Cancer Studies, D Floor, West Block, Queen's Medical Centre, University Hospital, Nottingham NG7 2UH, UK
Correspondence to: Dr AM Grabowska, E-mail: anna.grabowska@nottingham.ac.uk
Revised 5 December 2006; accepted 14 December 2006; published online 30 January 2007
Gastrin isoforms, acting through a variety of receptors, have proliferative and anti-apoptotic effects on gastrointestinal (GI) cancers. A small interfering RNA (siRNA) targeting the gastrin gene was used to investigate the role of endogenous gastrin in GI cancer cell survival. Downregulation of the gastrin gene in siRNA-transfected cells was measured using real-time reverse transcriptase-PCR. The most effective siRNA was tested in a panel of GI cancer cell lines at various concentrations and time points, and the effect on cell survival and apoptosis was measured using methyl thiazoyl tetrazolium (MTT) and caspase 3 activation assays. Gastrin siRNA reduced gene expression by more than 90% in a range of GI cancer cell lines. Downregulation of the gastrin gene was dose-dependent and effective over approximately 1 week in vitro. However, downregulation at the protein level was delayed by 3-4 days. Gastrin siRNA-transfected cells showed up to a 60% reduction in growth and up to a 50% increase in apoptosis compared with control siRNA-transfected cells. The effects were most marked in the cell line with the highest constitutive level of gastrin gene expression (human metastatic colon, C170HM2) and in epidermal growth factor (EGF)-treated cells as the gastrin promoter contains an EGF-response element, gERE. The ability of the siRNAs to reduce survival of these GI cell lines is further evidence of the importance of autocrine and/or intracrine gastrin loops in GI cancer, where expression of the gastrin gene and autonomous gastrin appears widespread.
Keywords: gastrin; gastrointestinal cancer; interfering RNA
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