Use of interfering RNA to investigate the role of endogenous gastrin in the survival of gastrointestinal cancer cells.
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.
Grabowska
A M
AM
Academic Unit of Cancer Studies, D Floor, West Block, Queen's Medical Centre, University Hospital, Nottingham NG7 2UH, UK. anna.grabowska@nottingham.ac.uk
Hughes
J
J
Watson
S A
SA
D017209:Apoptosis; D045744:Cell Line, Tumor; D002470:Cell Survival; D015536:Down-Regulation; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005755:Gastrins / Q000235:genetics* / Q000502:physiology*; D005770:Gastrointestinal Neoplasms / Q000473:pathology*; D006801:Humans; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology
D005755:Gastrins; D034741:RNA, Small Interfering; D004815:Epidermal Growth Factor