Snail Overexpression Induces An Epithelial To Mesenchymal Transition And Cancer Stem Cell-like Properties In Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma (OSCC) is the most frequent type of cancer in the oralcavity. Local invasiveness and distant metastasis are critical factors that contribute toOSCC-related deaths. Recently, a growing body of research suggests that theepithelial to mensenchymal transition (EMT) plays a critical role in the process. Thetranscriptional repressor Snail, correlated with the EMT by suppressing E-cadherinexpression.Objective：In this study, we construct a eukaryotic expression vector carrying Snailgene in order to detact the biological behavior of SCC9cells that overexpression ofSnail, then understand the role of Snail in EMT and cancer progression, and examinethe cancer stem cell like (CSC-like) properties induced by Snail in the SCC9cells.Methods：The eukaryotic expression vector pEGFP-N1-Snail was constructed. Thenthe pEGFP-N1and pEGFP-N1-Snail plasmids were purified and were transfectedinto the SCC9cells by Lipofectamine2000. At48h post-transfection, the media wassupplemented with400g/mL G418. Stable SCC9-pEGFP-N1(SCC9-N) andSCC9-pEGFP-N1-Snail (SCC9-S) cell lines were obtained and subcultured. Then, theexpression of E-cadherin,-catenin, Snail and vimentin were detected by usingquantitative real-time PCR, western blotting and immunofluorescence microscopy.Wound-healing, invasion and adhesion assays were performed to measure the cellinvasion and metastasis. Furthermore, the CSC-like characteristics in the SCC9-Scells was evaluated by flow cytometry and colony-forming assay.Results：The eukaryotic expression vector pEGFP-N1-Snail was constructed. SCC9 cells were transfected with an ampty vector or a vector encoding human Snail, StableSCC9-pEGFP-N1(SCC9-N) and SCC9-pEGFP-N1-Snail (SCC9-S) cell lines wereobtained. Overexpression of Snail induced SCC9cells to undergo the EMT, theSCC9-S cells showed typical fibroblast-like and spindle-shaped appearances, whereasthe SCC9-N cells showed robust cellular junctions with typical “cobblestone-shaped”and epithelial-like appearances, the epithelial markers E-cadherin and-catenin weredownregulated, the mesenchymal marker vimentin was upregulated, and associatedwith highly invasive and metastatic properties. Furthermore, the induction of theEMT promoted CSC-like characteristics in the SCC9-S cells, in which the cellsexhibited high invasion, but low proliferation. They did not migrate into the scratchedarea, presented a low percentage of cells in S phase, showed decrease in colonyformation, but have self-renewal ability that can form new colonies, and expresscancer stem cell-like markers. CD24expression was lower in the SCC9-S cells thanin the SCC9-N cells. A high level of CD44expression was assessed in the two celllines, but CD133expression was not detected in the two cell lines. Thus, the SCC9-Scells presented as CD133-negative and CD44high/CD24low, and the SCC9-N cellspresented as CD133-negative and CD44high/CD24+.Conclusions：In the present study, the eukaryotic expression vector pEGFP-N1-Snailwas constructed and the stable SCC9-S cells were abtained. Overexpression of Snailinduces the EMT and promotes CSC-like traits in the SCC9cells. These resultsfurther confirmed the role of Snail in oral cancer progression.