Flotillin-1 promotes EMT of gastric cancer via stabilizing Snail

Cell culture

The human gastric cancer cell line SGC-7901 was obtained from GeneChem (Shanghai, China), AGS was acquired from the Shanghai Institute for Biological Science. Both of the cell lines were cultured in RPMI/1640 medium (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Waltham, MA, USA) and incubated at 37 °C with 5% CO₂. HEK293T cell were purchased from American Type Culture Collection (ATCC) and grown in DMEM medium (Gibco, Waltham, MA, USA) plus with 10% FBS.

Plasmids and antibodies

Flotillin-1 and Snail were inserted into the pLVX vector. The Flotillin-1 shRNA sequences were cloned into the pSIH vector. The sequences of shRNAs are shown in Table S1.

Antibodies used in this study: anti-Flotillin-1 (#18634; Cell Signaling Technology, Danvers, MA, USA), anti-Snail (#3879; Cell Signaling Technology, Danvers, MA, USA), anti-E-cadherin (#3195; Cell Signaling Technology, Danvers, MA, USA), anti-N-cadherin (#13116; Cell Signaling Technology, Danvers, MA, USA), anti-Vimentin (#5741; Cell Signaling Technology, Danvers, MA, USA), anti-β-actin (A5441; Sigma-Aldrich, USA), anti-Flag (#14793; Cell Signaling Technology, Danvers, MA, USA), anti-HA (sc-53516; Santa Cruz Biotechnology, Dallas, TX, USA), Goat anti-Rabbit IgG (AS014; ABclonal, Hubei, China), Goat anti-mouse IgG (AS003; ABclonal, Hubei, China).

Generation of stable transfected cells

The stable transfected cells including Flotillin-1-overexpressing and -knockdown were generated by lentivirus. The lentivirus was generated using packaging vectors PSPAX2, pMD2G and Flotillin-1-overexpressing plasmid or Flotillin-1-shRNA plasmid. SGC-7901 and AGS cells were infected with indicated lentivirus and performed the stable transfected cell screening using puromycin. The third generation of stable transfected cells were used for subsequent experiments.

Quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from cells using TRIzol reagent and cDNA was prepared from total RNA using the Quantscript RT Kit following manufacturer’s instruction. qRT-PCR assay was performed using SYBR Green Mix following the manufacturer’s instruction. The primers used in qRT-PCR were shown in Table S2.

Immunoblotting assay

Cells were lysed by RIPA lysis buffer plus a cocktail of protease inhibitor. The protein concentration was detected using Bicinchoninic acid (BCA) assay. Total proteins (10 µg for each well) were separated by SDS-PAGE gel and transferred to Polyvinylidene difluoride (PVDF) membranes. The membrane was blocked with 5% fat-free milk for 1 h and incubated with primary antibodies overnight. Next day, the membrane was incubated with conjugated secondary antibody. The protein signal was visualized using ECL detection reagents.

Migration and invasion assay

Cell migration and invasion assay were performed using 24-well Transwell plate with or without coated Matrigel. Cells (5 × 10⁴ for each well) were plated into Boyden chambers with serum-free media. Media containing 10% FBS were added into the bottom chambers, and the cells were cultured for 24 h at 37 °C with 5% CO₂. Finally, the cells were fixed by methanol and stained with crystal violet.

Wound healing assay

Cells (2 × 10⁵ for each well) were plated into 6-well plates and scratched using a sterile pipette tip. The cells were washed with phosphate-buffered saline (PBS) and incubated in media containing 2% FBS. Images were obtained at 0, 24, 48, 72 h.

Immunoprecipitation assay

Cells were treated with 20 µg/ml MG132 for 6 h and lysed with RIPA lysis buffer plus a cocktail of protease inhibitor. For Flag-tagged proteins, cell lysates were incubated with anti-Flag M2 magnetic beads overnight. Finally, the immunoprecipitates were subjected to immunoblotting.

Ubiquitination assay

For ubiquitination assay, the cells were treated with MG132 for 6 h and lysed using RIPA lysis buffer plus a cocktail of protease inhibitor. The cell lysates were incubated with anti-Flag M2 magnetic beads overnight. The ubiquitin level of Flag-Snail was detected with anti-HA antibody.

Protein degradation analysis

For Snail protein degradation analysis, cells were treated with cycloheximide (CHX, 50 µg/ml) for 1, 2 h. The protein level of Snail was detected using immunoblotting assay.

Statistical analysis

Survival analysis was analyzed using a two-sided log-rank test, others were analyzed using unpaired t-test. Except for special instructions, all results were shown as mean ± SEM of three independent experiments. All statistical analyses were performed with Graphpad Prism. A p < 0.05 was considered significant.

Increased Flotillin-1 predicts a poor prognosis of gastric cancer patients

To identify the effect of Flotillin-1 expression level on the clinical prognosis of gastric cancer, we analyzed the data from The Cancer Genome Atlas (TCGA). A Flotillin-1 amplification occurs in several solid tumors including gastric cancer (Fig. 1A). Compared to normal tissues, the expression level of Flotillin-1 was significantly higher in gastric cancer samples (Fig. 1B). Gastric cancer patients with high Flotillin-1 expression level correlated with a worse survival overall survival, advanced TNM stage and distant metastasis (Figs. 1C–1E). Together, those results shown that Flotillin-1 can participate in the development of gastric cancer.

Overexpression of Flotillin-1 promotes gastric cancer metastasis

To identify the function of Flotillin-1 in gastric cancer, we overexpressed Flotillin-1 in two gastric cancer cell lines SGC-7901 and AGS. The overexpression efficiency was confirmed by qRT-PCR and immunoblotting (Figs. 2A–2D). To examine the role of Flotillin-1 in gastric cancer metastasis, we performed Transwell and wound healing assays to detect the abilities of migration and invasion. Overexpression of Flotillin-1 not only promoted the migration and invasion of SGC-7901 cells, but also increased the wound healing ability of gastric cancer cells (Figs. 2E–2F). In addition, we obtained the same results in AGS cells (Figs. 2G–2H). Taken together, the results show that overexpression of Flotillin-1 promotes gastric cancer cell migration and invasion and suggested that Flotillin-1 might have a role in cancer metastasis.

Knockdown of Flotillin-1 inhibits gastric cancer metastasis

To further study the role of Flotillin-1 in gastric cancer metastasis, we knocked down Flotillin-1 in SGC-7901 and AGS cells using shRNA. The knockdown efficiency was confirmed by qRT-PCR and immunoblotting (Figs. 3A–3D). We performed Transwell and wound healing assays to detect the cell migration and invasion abilities of Flotillin-1-knockdown and control gastric cancer cells. As expected, knockdown of Flotillin-1 inhibited the migration, invasion and wound healing abilities of SGC-7901 and AGS cells (Figs. 3E–3H). Thus, the results show that knockdown of Flotillin-1 inhibits gastric cancer cell migration and invasion and suggested that Flotillin-1 might have a role in cancer metastasis.

Flotillin-1 promotes gastric cancer metastasis through inducing EMT

The initiation of metastasis requires invasion, which is achieved through EMT. To identify whether Flotillin-1 promotes gastric cancer metastasis through EMT process. We detected the expression of several EMT markers including epithelial marker E-cadherin and mesenchymal markers N-cadherin, Vimentin and Snail. Overexpression of Flotillin-1 downregulated the protein and mRNA levels of E-cadherin, and upregulated the N-cadherin, Vimentin and Snail levels (Figs. 4A–4D). In contrast, knockdown of Flotillin-1 increased the expression of E-cadherin and inhibited the expression of N-cadherin, Vimentin and Snail (Figs. 4E–4H). Moreover, we found that Flotillin-1 regulated the protein level of Snail, but has no effect on the mRNA level. Taken together, those results determined that Flotillin-1 promotes the EMT process in gastric cancer cells.

Flotillin-1 promotes EMT of gastric cancer via stabilizing Snail

Snail, as an EMT-transcription factor, has been confirmed to play an integral role throughout EMT of all types. In addition, Snail is a labile protein and degraded by ubiquitin-proteasome system, and Flotillin-1 regulates the protein expression of Snail but no effect on mRNA level. Thus, we speculated that Flotillin-1 promoted the EMT process through increasing the protein stability of Snail. To identify the hypothesis, we performed immunoprecipitation and ubiquitination assays. As expected, Flotillin-1 interacted with Snail (Fig. 5A) and decreased the ubiquitination level of Snail (Fig. 5B). In addition, Flotillin-1 inhibited the degradation of Snail in gastric cancer cells (Figs. 5C–5D). Taken together, the results shown that Flotillin-1 decreased the ubiquitination level of Snail and inhibited its degradation.