Flotillin-1 promotes EMT of gastric cancer via stabilizing Snail

Figures

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  Figure 1. Increased Flotillin-1 predicts a poor prognosis of gastric cancer patients. (A) A histogram shows the amplification frequency of Flotillin in 23 tumor types. The data were taken from TCGA PanCancer Altas studies. (B) The expression of Flotillin-1 in gastric cancer tissues and normal samples. T represents tumor tissues (n = 408), N represents normal tissues (n = 36). (C) Kaplan-Meier plot showing the overall survival of 875 gastric cancer patients stratified by Flotillin-1 expression level. (D–E) Comparison between the proportion of gastric cancer patients with different Flotillin-1 expression level in T stage (D) and distant metastasis (E). *p < 0.05, **p < 0.01, p value was calculated with t test, except survival analysis was analyzed using a two-sided log-rank test.

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  Figure 2. Overexpression of Flotillin-1 promotes gastric cancer metastasis. (A–B) The mRNA (A) and protein (B) levels of Flotillin-1 in SGC-7901 cells overexpressing Flotillin-1. (C–D) The mRNA (C) and protein (D) levels of Flotillin-1 in AGS cells overexpressing Flotillin-1. (E) The migration and invasion abilities of SGC-7901 cells overexpressing Flotillin-1 were detected by Transwell assay. Scale bars, 500 µm. (F) The migration ability of SGC-7901 cells overexpressing Flotillin-1 was examined by wound healing assay. Scale bars, 500 µm. (G) The migration and invasion abilities of AGS cells overexpressing Flotillin-1 were detected by Transwell assay. Scale bars, 500 µm. (H) The migration ability of SGC-7901 cells overexpressing Flotillin-1 was examined by wound healing assay. Scale bars, 500 µm. **p < 0.01, ***p < 0.001, ****p < 0.0001, p value was calculated with t test, compared to control group. All results were shown as mean ± SEM of three independent experiments.

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  Figure 3. Knockdown of Flotillin-1 inhibits gastric cancer metastasis. (A–B) The mRNA (A) and protein (B) levels of Flotillin-1 in Flotillin-1-knockdown (using shRNA targeting Flotillin-1 including shFlotillin-1-1 and shFlotillin-1-2) SGC-7901 cells. (C–D) The mRNA (left) and protein (right) levels of Flotillin-1 in Flotillin-1-knockdown AGS cells. (E) The migration and invasion abilities of Flotillin-1-knockdown SGC-7901 cells were detected by Transwell assay. Scale bars, 500 µm. (F) The migration ability of Flotillin-1-knockdown SGC-7901 cells was examined by wound healing assay. Scale bars, 500 µm. (G) The migration and invasion abilities of Flotillin-1-knockdown AGS cells were detected by Transwell assay. Scale bars, 500 µm. (H) The migration ability of Flotillin-1-knockdown AGS cells was examined by wound healing assay. Scale bars, 500 µm. **p < 0.01, ***p < 0.001, ****p < 0.0001, p value was calculated with t test, compared to control group. All results were shown as mean ± SEM of three independent experiments.

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  Figure 4. Flotillin-1 promotes gastric cancer metastasis through inducing EMT. (A–B) The protein expression levels of E-cadherin, N-cadherin, Vimentin and Snail in Flotillin-1 overexpressing SGC-7901 (A) and AGS (B) cells. (C–D) The mRNA levels of E-cadherin, N-cadherin, Vimentin and Snail in Flotillin-1 overexpressing SGC-7901 (C) and AGS (D) cells. (E–F) The protein expression levels of E-cadherin, N-cadherin, Vimentin and Snail in Flotillin-1-knockdown SGC-7901 (E) and AGS (F) cells. (G–H) The mRNA levels of E-cadherin, N-cadherin, Vimentin and Snail in Flotillin-1-knockdown SGC-7901 (G) and AGS (H) cells. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, p value was calculated with t test, compared to control group. All results were shown as mean ± SEM of three independent experiments.

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  Figure 5. Flotillin-1 promotes EMT of gastric cancer via stabilizing Snail. (A) Snail and Flag-Flotillin-1 were co-transfected into HEK293T cells. The cells were treated with MG132 for six hours and subjected into immunoprecipitation assay with anti-Flag antibody to pulldown Flag-Flotillin-1 immunoprecipitates. (B) HA-ubiquitin, Flag-Snail and Flotillin-1 were co-transfected into HEK293T cells. After MG132 treatment, the cells were subjected into ubiquitination assay with anti-Flag antibody. The ubiquitination level of Snail was detected using anti-HA antibody. (C) Flotillin-1-knockdown and control SGC-7901 cells were treated with CHX as indicated. The degradation of Snail was detected by immunoblotting. (D) Flotillin-1-knockdown and control AGS cells were treated with CHX as indicated. The degradation of Snail was detected by immunoblotting. *p < 0.05, p value was calculated with t test, compared to control group at 2 h point.