Overexpression of stathmin1 in the diffuse type of gastric cancer and its roles in proliferation and migration of gastric cancer cells

Abstract

Background: Stathmin1 is a microtubule-regulating protein that has an important role in the assembly and disassembly of the mitotic spindle. The roles of stathmin1 in carcinogenesis of various cancers, including prostate and breast cancer, have been explored. However, its expression and roles in gastric cancer have not yet been described.

Methods: Stathmin1 expression in paraffin-embedded tissue sections from 226 patients was analysed by immunohistochemistry. Roles of stathmin1 were studied using a specific small interfering RNA (siRNA).

Results: The expression of stathmin1 was positively correlated with lymph node metastasis, TNM stages and vascular invasion, and negatively with recurrence-free survival, in the diffuse type of gastric cancer. The median recurrence-free survival in patients with a negative and positive expression of stathmin1 was 17.0 and 7.0 months, respectively (P=0.009). When the expression of stathmin1 was knocked down using siRNA, the proliferation, migration and invasion of poorly differentiated gastric cancer cells in vitro were significantly inhibited. Moreover, stathmin1 siRNA transfection significantly slowed the growth of xenografts in nude mice.

Conclusion: These results suggest that stathmin1 can be a good prognostic factor for recurrence-free survival rate and is a therapeutic target in diffuse-type gastric cancer.

Figure 1

Immunohistochemical staining of stathmin1 in oral and gastric cancer sections. Anti-stathmin1 antibody was used for immunohistochemical staining in human oral and gastric cancer tissues as described in ‘Materials and Methods’ section. The invading oral cancer cells were positive for stathmin1 protein in the cytoplasm. The normal gastric mucosal cells were either negative or very weakly positive for stathmin1 protein in the cytoplasm. The invading gastric cancer cells were positive for stathmin1 protein. Scale bar, 200 μm.

Figure 2

Correlationship between the stathmin1 expression level and clinicopathological characteristics. Data of stathmin-positive patients with diffuse-type gastric cancer are presented. (A) The state of stathmin1 protein expression in patients with lymph node metastasis in diffuse type gastric cancer (n=17) and in patients without metastasis (n=7). Stathmin1 protein expression in patients with lymph node metastasis is significantly higher than in patients without lymph node metastasis (^*P<0.05, Mann–Whitney U-test). (B) The state of stathmin1 protein expression in patients with early stages (I and II) of diffuse type gastric cancer (n=9) and patients with advanced stages (III and IV) of diffuse-type gastric cancer (n=15). Stathmin1 protein expression in advanced stages is significantly higher than that in early stages (^**P<0.01, Mann–Whitney U-test). (C) The state of stathmin1 protein expression in patients with vascular invasion in diffuse-type gastric cancer (n=14) and in patients without vascular invasion (n=10). Stathmin1 expression in patients with vascular invasion is significantly higher than in patients without vascular invasion (^**P<0.01, Mann–Whitney U-test).

Figure 3

Kaplan–Meier curves for recurrence-free survival according to stathmin1 overexpression in patients with gastric cancer ((A) diffuse type+intestinal type, P<0.05; (B) diffuse type only, P<0.01). When stathmin1 overexpression was observed, the recurrence-free survival rate decreased significantly, especially in patients with diffuse-type gastric cancer.

Figure 4

Stathmin1 was specifically downregulated by stathmin1siRNA. Cells were transfected with scrambled siRNA or stathmin1 siRNA. (A) Two days later, SNU638 and SNU16 cells were collected and stathmin1 protein levels were detected by western blotting. β-actin expression was monitored for normalisation. (B) SNU638 cells were collected and stathmin1 mRNA levels were examined by real-time PCR. Values are expressed as the percentage of control, which was defined as 100% (n=6). Results were analysed by one-way ANOVA, followed by Tukey's multiple comparison test. ^*P<0.01 vs control. mRNA, messenger RNA; SCR, scrambled; siRNA, small interfering RNA.

Figure 5

Effect of stathmin1 silencing on proliferation of gastric cancer cells. SNU638 (A) or SNU16 (B) cells were transfected with SCR siRNA or stathmin1 (STMN1) siRNA. After 5 days of incubation, cell proliferation was evaluated in WST assay. Data are expressed as percentage change (means±s.d.) compared with controls and represent four independent experiments. (^*P<0.05, ^**P<0.01 vs SCR siRNA, one-way ANOVA followed by Tukey's multiple comparison). ANOVA, one-way analysis of variance; SCR, scrambled; siRNA, small interfering RNA.

Figure 6

Effect of stathmin1 silencing on the migration and invasion of gastric cancer cells. (A) Cell migration was evaluated in the Boyden migration assay two days after SNU638 cells were transfected with scrambled (SCR) small interfering RNA (siRNA) or stathmin1 siRNA. (B) Cell invasion was evaluated in the Matrigel invasion assay as described in the ‘Materials and Methods’ section. Data are expressed as percentage change (means±s.d.) compared with controls and represent four independent experiments. (^*P<0.01 vs SCR siRNA, one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison). Representative microscopic images were presented in the upper panel of each assay graph. ANOVA, one-way analysis of variance; SCR, scrambled; siRNA, small interfering RNA.

Figure 7

Effect of stathmin1 silencing on in vivo tumour growth. (A) SNU638 cells were transfected with scrambled (SCR) small interfering RNA (siRNA) or stathmin1 (STMN1) siRNA, and nude mice were inoculated subcutaneously with 2 × 10⁶ cells at two sites per mouse. The tumour mass (xenograft) volume was measured every week from week 3 to week 7. Data are expressed as the means±s.d. and represent four independent experiments. (^*P<0.05, ^**P<0.01 vs SCR siRNA, one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison) (B) The tumour masses (xenograft) were dissected out 7 weeks later and presented. ANOVA, one-way analysis of variance; SCR, scrambled; siRNA, small interfering RNA.