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. 2011 Feb;3(2):209-18.
Epub 2011 Feb 6.

A microRNA contribution to aberrant Ras activation in gastric cancer

Emily Ky Lam, Xian Wang, Vivian Y Shin, Shengjie Zhang, Helen Morrison, Jie Sun, Enders Ko Ng, Jun Yu, Hongchuan Jin

A microRNA contribution to aberrant Ras activation in gastric cancer

Emily Ky Lam et al. Am J Transl Res. 2011 Feb.

Abstract

Oncogenic Ras mutations are rare in gastric cancer, indicating that other mechanisms may be responsible for aberrant Ras activation in this type of cancer. Ezrin is critical to Ras activation by remodeling cortical actin cy-toskeleton. In this study, we aimed to illustrate the relevance and regulation of ezrin in gastric cancer. Ezrin was upregulated in gastric cancer cells. Ezrin siRNA inhibited Ras activation, cell growth and cell migration. Ezrin overex-pression was correlated with a poor outcome of gastric cancer patients (n=150, p<0.01). Cox regression analysis revealed a significant value of ezrin expression in prognosis prediction of gastric cancer (relative risk: 2.37, 95% confidence interval: 1.24-4.56, p<0.01). MiR-204, which was predicted to target ezrin, was downregulated in gastric cancer cells and gastric carcinomas (n=22, p<0.01). MiR-204 inhibited ezrin expression, Ras activation, cell growth and cell migration. Importantly, miR-204 suppressed the expression of luciferase controlled by wild-type but not mutated ezrin 3'-UTR. In conclusion, ezrin is important to Ras activation in gastric cancer. Its upregulation is an independent prognosis prediction factor for gastric cancer. By contributing to ezrin upregulation, miR-204 downregulation represents a novel mechanism for aberrant Ras activation in gastric carcinogenesis.

Keywords: Ezrin; Ras; gastric cancer; miR-204.

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Figures

Figure 1
Figure 1
Effect of ezrin knock-down on Ras activation, cell growth and cell migration. A. Ezrin expression in a panel of gastric cancer cell lines was determined by immunoblotting. Normal (non-tumour, non-inflammation) stomach tissue was used as the reference. B. Ras activity and Erk1/2 phosphorylation in gastric cancer cells (NCI-N87) before and after ezrin knockdown were determined by immunoblotting. Active Ras indicates Ras-GTP pulled down by Raf-RBD. MTS assay (C. NCI-N87 and AGS cells) and cell migration assay (D. AGS cells) were used to determine the effect of ezrin knock-down on cell growth and cell migration, respectively. A representative experiment in triplicate is shown as mean±SD. The asterisks indicate a statistically significant difference (Student's t-test, p<0.05).
Figure 2
Figure 2
Association of ezrin expression with patient survival. Association of ezrin expression with patient survival (A. all four stages and B. stage I and II) was analyzed by Kaplan-Meier survival curve. Ezrin expression was determined by immunohistochemistry. The p values were calculated by log-rank test.
Figure 3
Figure 3
Downregulation of miR-204 in gastric cancer. Expression of miR-204 in gastric cancer cell lines (A) and primary tissues (B) was determined by quantitative real-time RT-PCR. Normal (non-tumour, non-inflammation) stomach tissue was used as the reference in A. Wilcoxon matched pair test was used in B (n=32, p<0.01) to analyze the difference in the expression of miR-204 in gastric carcinoma tissues and adjacent non-tumour tissues.
Figure 4
Figure 4
Direct targeting of ezrin expression by miR-204. Predicted interaction of miR-204 (grey) with ezrin 3'-UTR (black) is shown in A. B. Ezrin expression in NCI-N87 cells before and after miR-204 re-expression was determined by immunoblotting. The schematic of microRNA luciferase reporter construct is shown in C. WT: wild type; MT: mutated type. The seed sequence is underlined. D. Expression of luciferase in the presence of miR-204 or control oligonucleotides was determined by the luciferase reporter assay. A representative experiment is shown as mean±SD. The asterisk indicates a statistically significant difference (Student's t-test, p<0.05).
Figure 5
Figure 5
Effect of miR-204 on Ras activation, cell growth and cell migration. A. Ras activity and Erk1/2 phosphorylation in gastric cancer cells (NCI-N87) before and after miR -204 re-expression were determined as in Figure 1B. The effect of miR-204 on cell growth (B) and cell migration (C) was determined as in Figures 1C and 1D, respectively. A representative experiment in triplicate is shown as mean±SD. The asterisks indicate a statistically significant difference (Student's t-test, p<0.05).
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