Use of interfering RNA to investigate the role of endogenous gastrin in the survival of gastrointestinal cancer cells

Abstract

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.

Figure 1

(A) siRNA transfection efficiency. PAN-1 cells transfected with a TAMRA-labelled gastrin siRNA 24 h post-transfection (× 64 magnification). The siRNA is associated with vesicle-like structures in the cytoplasm of the cells. (B) Expression of the gastrin gene in cells transfected with different gastrin siRNAs. PAN-1 cells were transfected with six different gastrin siRNAs and gastrin expression was measured by real-time PCR. Expression relative to cells transfected with the scrambled control siRNA (scrtg5) and the housekeeping gene, HPRT, is shown. Error bars indicate the 95% confidence intervals. The relative position of each siRNA within the gastrin sequence is indicated.

Figure 2

Effectiveness of gastrin siRNA in a range of GI cancer cell lines. (A) Relative expression of gastrin in four cell lines (PAN-1, HCT116, MGLVA1 and C170HM2) is shown using the 2^−ΔCt formula. A significant difference in expression compared with that in PAN-1 (P<0.001) is indicated by ^*. (B) The downregulation of gastrin gene expression in cells treated with the tg8 gastrin siRNA is shown relative to cells treated with the control scrtg8 siRNA using the 2^−ΔΔCt formula. (C) Gene expression in PAN-1 cells on d2 after transfection with gastrin (tg8) or control (scrtg8) siRNA and 24 h treatment with or without 10 μg ml⁻¹ EGF. Data are expressed relative to the housekeeping gene, HPRT. Significant differences (P<0.001) are indicated by ^*. Error bars indicate the 95% confidence intervals.

Figure 3

Concentration-dependent effects of the gastrin siRNA and longevity of downregulation of the gastrin gene. (A) Gastrin gene expression in HCT116 cells transfected with a range of concentrations of gastrin (tg8) siRNA, expressed relative to the housekeeping gene, HPRT, and cells transfected with the same concentration of the control (scrtg8) siRNA. Error bars indicate the 95% confidence intervals. (B) Gene expression in PAN-1 cells at d1, d4, d7 or d11 after transfection with 0.5 or 0.05 μg gastrin (tg8) or control (scrtg8) siRNA. Data are expressed relative to the control siRNA and the housekeeping gene, HPRT. Error bars indicate the 95% confidence intervals.

Figure 4

Absence of induction of the innate interferon response by gastrin siRNA. Gastrin, OAS and STAT1 gene expression in PAN-1 cells treated with gastrin (tg8), control (scrtg8) siRNA or no siRNA (mock transfected). Error bars indicate the 95% confidence intervals.

Figure 5

Downregulation of GFP-tagged and endogenous gastrin protein expression by gastrin siRNA. Cells transfected with (A) a GFP-tagged gastrin plasmid and treated with control scrtg8 siRNA or (B) gastrin tg8 siRNA are shown 24 h after transfection. The percentage of positive cells is indicated and there was a significant difference (P<0.0001) between the two treatments. Cells transfected with (C) scrtg8 or (D) gastrin tg8 siRNA were immunostained for endogenous gastrin expression using an anti-C-terminal antibody 72 h after transfection. Three fields are shown for each treatment.

Figure 6

Survival of gastrin siRNA-transfected cells in the presence and absence of serum. Growth of PAN-1 cells transfected with gastrin tg8 siRNA or the control scrtg8 siRNA in serum containing 10% or serum-free medium over 8 days following transfection. At d3 and d6, there was significantly lower survival in the tg8- treated cells than the scrtg8-treated cells (P=0.004 and 0.001 for serum and no serum, respectively, at d3, and P=0.06 and 0.02, respectively, at d6).

Figure 7

Survival of gastrin siRNA-transfected cancer cells. Survival of (A) PAN-1, (B) C170HM2, (C) HCT116 and (D) MGLVA1 cells in serum-free medium with or without the addition of 10 μg ml⁻¹ EGF following transfection with gastrin (tg8) or control (scrtg8) siRNAs. Growth of tg8-transfected cells is shown as a percentage of scrtg8-treated cells. Significant differences (P<0.05) are indicated by ^*.

Figure 8

Apoptosis in gastrin siRNA-transfected cancer cells. Proportion of apoptotic cells at d4 following treatment with gastrin (tg8) or the control (scrtg8) siRNA in the presence or absence of 10 μg ml⁻¹ EGF. Apoptosis is shown relative to scrtg8-treated cells. Significant differences (P<0.05) are indicated by ^*. (A) PAN-1, (B) C170HM2, (C) HCT116 and (D) MGLVA1.

Figure 9

Growth of siRNA-transfected cells in the presence of gastrin peptides and a CCK2-R antagonist, (A) the growth of gastrin (tg8) or control (scrtg8) siRNA-transfected C170HM2 cells treated with 10 nM amidated (G17) or glycine-extended (Gly-G17) gastrin. The percentage increase in growth compared with transfected cells alone is indicated. There was a significant increase in the growth of peptide-treated gastrin siRNA-transfected cells compared with control siRNA-transfected cells (P<0.001 for both G17 and GlyG17, indicated by ^*). (B) The growth of gastrin (tg8) or control (scrtg8) siRNA-transfected Pan1 and C170HM2 cells treated with 500 nM YM022 is shown. There was a significant further reduction in cell survival in tg8-transfected Pan1 cells following treatment with YM022 (41%, P=0.003) is shown. There was a 23% reduction in cell survival in C170HM2 cells but this did not reach significance.