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Claudin6 Suppresses Migration and Invasion via Blocking Smads/Snail/MMP2/9 Pathway in MCF-7 and SKBR-3 Cell Lines

Ting Liu, Libo Jiang, Qingyang Bai, Shuqin Wu, Xiuwen Yu, Tian Wu, Junping Wang, Xiaojie Zhang, Hongmei Li, Kun Zhao, Liping Wang

Background: We have previously reported that CLDN6-mediated SB431542 suppresses EMT, migration and invasion in breast cancer cells, but the mechanisms need to be further studied. Our previous experiments verified that SB431542 a SMADs signaling pathway inhibitor inhibits invasion, migration and EMT, and down-regulates Snail, MMP2 and MMP9 expression in breast cancer cells. Knocking down CLDN6 reversed the effects of SB431542. In order to further study the mechanisms responsible for this effect, the following experiments were carried out.

Methods: 1- CLDN6 was knocked down by shRNA in MCF-7 and SKBR-3 cell lines, which had been pretreated with SB431542, The cells were incubated with MMP2/9 Inhibitor I selective antagonists of MMP-2/MMP-9. 2- Snail was knocked down by shRNA in MCF-7 and SKBR-3 cell lines. The levels of MMP2 and MMP9 were examined by RT-PCR and Western blot. The expression of E-cadherin and N-cadherin were analyzed by Western blot and Immunofluorescence Microscopy. Vimentin expression was detected by Western blot. Migration and invasion were analyzed by Wound Healing Assay and Matrigel Invasion Assay.

Results: MMP2/9 Inhibitor I reversed the effects of Knocking down CLDN6 on downregulation of E-cadherin, upregulation of N-cadherin and Vimentin, facilitation of migration and invasion. In MCF-7-shSnail and SKBR-3-shSnail cells, migration and invasion were inhibited, E-cadherin was up regulated, MMP2, MMP9, N-cadherin and Vimentin were downregulated.

Conclusions: The experiment demonstrates that Claudin6 suppresses epithelial-mesenchymal transition, migration and invasion via blocking SMADs/Snail/MMP2/9 pathway in MCF-7 and SKBR-3 cell lines.