Snail-induced epithelial-mesenchymal transition in gastric carcinoma cells and generation of cancer stem cell characteristics

Genet Mol Res. 2016 Aug 29;15(3). doi: 10.4238/gmr.15038510.

Abstract

Biological changes in Snail-overexpressed SGC7901 cells were studied by establishing a pEGFP-C1-Snail carrier. The significance of Snail in epithelial-mesenchymal transition (EMT) as well as the invasion and metastatic capacity of gastric cancer cells was also discussed; moreover, we attempted to verify the probable cancer stem cell characteristics of Snail-overexpressed cells. A pEGFP-C1-Snail eukaryotic expression plasmid was constructed and pEGFP-C1(-) and pEGFP-C1-Snail plasmids were extracted and transfected into SGC7901 cells using Lipofectamine 2000. Stably expressed SGC7901-N [control group containing pEGFP-C1(-)] and SGC7901-S (test group containing pEGFP-C1-Snail) cells were screened using a G418 resistance medium. Snail, E-cadherin, b-catenin, vimentin, and fibronectin gene and protein expressions were detected by real-time quantitative PCR, western blot, and immunofluorescence. Cell invasion and metastasis were tested by scratch test, invasion assay, and an adhesion experiment. The positive rate of aldehyde dehydrogenase-1 (ALDH-1) expression was analyzed by flow cytometry. The results indicated the occurrence of EMT, accompanied by morphological changes in the cells and a weakening of the cell adhesion capacity. We also observed a decrease in the expression of epithelial markers E-cadherin and b-catenin and an increase in mesenchymal (Snail and vimentin) marker expression. Moreover, the cells showed increased invasiveness and metastatic capacity, and decreased proliferative ability. Moreover, the Snail-treated SGC7901 cells moved towards the scratch and produced fewer clones compared to the control cells. Owing to its capacity for self-renewal, SGC7901-S cells produced new clones and expressed ALDH-1. Therefore, we concluded that Snail overexpression induced EMT and endowed cells with tumor stem cell characteristics.

MeSH terms

  • Aldehyde Dehydrogenase 1 Family
  • Antigens, CD
  • Cadherins / genetics
  • Cadherins / metabolism
  • Cell Adhesion
  • Cell Line, Tumor
  • Cell Movement
  • Cell Proliferation
  • Epithelial Cells / metabolism*
  • Epithelial Cells / pathology
  • Epithelial-Mesenchymal Transition / genetics*
  • Fibronectins / genetics
  • Fibronectins / metabolism
  • Gastric Mucosa / metabolism
  • Gastric Mucosa / pathology
  • Gene Expression Regulation, Neoplastic*
  • Genes, Reporter
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Isoenzymes / genetics
  • Isoenzymes / metabolism
  • Neoplastic Stem Cells / metabolism*
  • Neoplastic Stem Cells / pathology
  • Plasmids / chemistry
  • Plasmids / metabolism
  • Retinal Dehydrogenase / genetics
  • Retinal Dehydrogenase / metabolism
  • Signal Transduction
  • Snail Family Transcription Factors / genetics*
  • Snail Family Transcription Factors / metabolism
  • Transfection
  • Transgenes
  • Vimentin / genetics
  • Vimentin / metabolism
  • beta Catenin / genetics
  • beta Catenin / metabolism

Substances

  • Antigens, CD
  • CDH1 protein, human
  • CTNNB1 protein, human
  • Cadherins
  • Fibronectins
  • Isoenzymes
  • SNAI1 protein, human
  • Snail Family Transcription Factors
  • Vimentin
  • beta Catenin
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • Aldehyde Dehydrogenase 1 Family
  • ALDH1A1 protein, human
  • Retinal Dehydrogenase