Effects of c-Ski oncogene on the activation of cancer-associated fibroblasts

Abstract:

Objective：To research the effect of c-Ski oncogene on the activation of breast cancer-associated fibroblasts（CAFs）. Methods：The c-Ski gene was cloned into the vector pBABE-puro to construct the expression vector pBABE-puro-Ski then was transfected into normal fibroblasts（NFs）；the stable transfected fibroblasts were selected by puromycin. Western blot was used to ana－lyze the expression of c-Ski，α-smooth muscle actin（α-SMA） and fibroblast activation protein（FAP） after the transfection. MTT as－say，flow cytometry，wound-healing experiment and Transwell assay were used to study the differences of cell proliferation，migration and invasion before and after the transfection. Conditioned media were collected and used to study the effect of fibroblasts on the in－vasion of MDA-MB-231 cells by Transwell assay. Results：The recombinant plasmid pBABE-puro-Ski was successfully constructed. Western blot showed that the expressions of c-Ski，α-SMA and FAP were higher in cells transfected with pBABE-puro-Ski than in those untransfected cells（P=0.000，P=0.001，P=0.002）. MTT showed that the proliferation of pBABE-puro-Ski transfected cells was higher than that of untransfected cells（P=0.011）. FCM showed that the S phase of pBABE-puro-Ski transfected cells（38.58±1.28） was significantly higher than that of empty vector transfected（25.05±0.52） or untransfected cells（26.01±0.86）（P=0.000）. Wound-healing experiment and Transwell assay revealed enhanced migration and invasion of the pBABE-puro-Ski transfected cells com－pared with those of the other two groups. Transwell assays showed that pBABE-puro-Ski transfected cells could promote the invasion of MDA-MB-231 cells. Conclusion：Overexpression of c-Ski could stimulate the activation of breast CAFs and activated CAFs further promote the invasion of MDA-MB-231 cells.