CLDN6-mediates SB431542 action through MMPs to regulate the invasion migration and EMT of breast cancer cells

摘要

Our previous research confirmed the repression of SMADs signaling pathway inhibits the invasion, migration, and EMT in breast cancer MCF-7 and SKBR-3 cell lines by DNMT1 up-regulating CLDN6, but the mechanism is unclear. Western blot was performed to detect the expression of SMAD2, SMAD3, P-SMAD2, and P-SMAD3. Then RT-PCR was carried out to examine the expression of tight junctions and cell adhesion molecule E-cadherin. According to the gene sequence of Claudin6, shRNA was linked with the green fluorescent protein-expressing eukaryotic expression vector pGC silencer TMΜ6/Neo/GFP, and it was transfected into breast cancer MCF-7 cells and SKBR-3 cells. RT-PCR and western blot were applied to verify the Claudin6 gene-silencing effect. We observed cellular morphology with inverted microscope, analyzed the capacity for clone formation, and detected transepithelial electrical resistance. The level of MMP2, and MMP9 in the cells treated with or without SB431542 and MCF-7-shGFP, MCF-7-shClaudin-6, SKBR-3-shGFP, and SKBR-3-shClaudin-6 cells pretreated with SB431542 were examined by RT-PCR and western blot. The expressions of Claudin-6, occludin, and cell adhesion molecule E-cadherin were enhanced by SB431542. SB431542 transformed mesenchymal cell morphology into epithelial cell morphology, inhibited capacity for clone formation, increased transepithelial electrical resistance, and downregulated the expression of MMP2 and MMP9. Knock down of Claudin6 can abolish SB431542 effects. We conclude that Claudin6 mediates the effects of SB431542 on the biologic phenotypes of the breast cancer cells we studied. We speculate Claudin6-mediated the SB431542 inhibition of invasion, migration, and EMT in breast cancer cells via MMP2/9.