CLDN6 Inhibits Migration And Invasion Of Breast Cancer Cells MCF-7 By Regulating IL-8/CXCR2 Signaling Pathway

Claudins(CLDNs)are basic proteins that form the tight junctions(TJs)and maintain the function of TJs.The abnormal expression of CLDNs plays a very important role in tumorigenesis,development and metastasis.Among them,Claudin6(CLDN6)is one of the 27 Claudin CLDNs family members with four transmembrane regions.The carboxyl terminal located in the cytoplasm has a PDZ-binding domain that binds to signal proteins and cytoskeletal proteins and participates in cells.Responses to external signals and intracellular signaling processes affect the overall function of cells through downstream molecular elements and play a role in tumor metastasis.In our earlier work,we found that CLDN6 expression was down-regulated in breast cancer cells and tissues,and overexpression of CLDN6 significantly inhibited the migration and invasion of breast cancer cells.The results of pre-experiment showed that after overexpression of CLDN6,fibroactin F-actin in breast cancer cells was depolymerized in large quantities,and there was less pseudopodia on the edge of breast cancer cells.A large number of cytoskeletons were observed in the nucleus and around the nucleus(transmission electron microscope).It is suggested that CLDN6 inhibits tumor cell migration and invasion by cytoskeleton rearrangement.The most important role of cytoskeleton is to maintain cell morphology and structure and participate in cell movement.F-actin,which distributes in the pseudopodia,controls the contraction of the pseudopodia by dissociation and polymerization,and then regulates the movement of the cells.In the process of tumor cell metastasis,the continuous aggregation,depolymerization and rearrangement of microfilaments drive the migration of tumor cells.Studies have shown that there are many signal pathways involved in regulating cytoskeleton affecting tumor cell migration and invasion.Among them,IL-8 is a CXC type inflammatory chemokine,which has been reported to be related to cytoskeleton.IL-8 binds to receptor CXCR2,up-regulates the expression of tyrosine residue phosphorylation by integrin-activated Rho GTP enzyme,and activates the phosphorylation of cortical protein Cortactinin complex to regulate F-actin polymerization,resulting in cytoskeleton rearrangement affecting migration and invasion.In conclusion,IL-8/CXCR2 signaling pathway regulates F-actin polymerization through integrin-activated FAK phosphorylation,resulting in cytoskeleton rearrangement affecting migration and invasion.As a chemokine,IL-8 is regulated by a variety of signal molecules.It has been shown that IL-8 binds to the closed microcyclic protein(ZO-1)through the PDZ domain and plays a role in signal transduction during tumor cell metastasis.ZO-1 is a tightly linked protein.Involved in the regulation of cell substance transport and maintenance of epithelial cell polarity ZO-1 and transcription factor AP-1,NF-κB,IL-8 promoter is involved in the regulation of IL-8 expression in breast cancer cells.We speculate that CLDN6 may influence cytoskeleton rearrangement through IL-8/CXCR2 signaling pathway to inhibit the migration and invasion of breast cancer cells.Objective:To explore the mechanism of CLDN6 affecting the migration and invasion of breast cancer MCF-7 cells by regulating the IL-8/CXCR2 signaling pathway,and to provide some experimental evidence for the treatment of breast cancer targeting CLDN6.Method:1.RT-PCR Western-Blot technique was used to identify the expression of CLDN6 in MCF-7 of breast cancer cells.2.The expression of IL-8 in MCF7/CLDN6 cells was detected by polymerase chain reaction(RT-PCR).3.Selection of optimal concentration and time of IL-8 by scratch experiment;After the MCF7/CLDN6 cells were treated with the best concentration and time of IL-8,the MCF7/CLDN6 cells were treated with 0.1 BSA as control.4.The effect of IL-8 on the morphology of MCF7/CLDN6 cells was observed under microscope,and the cytoskeleton of MCF7/CLDN6 cells was observed byfluorescence staining and transmission electron microscopy(TEM).5.The effect of IL-8 on the migration and invasion of MCF7/CLDN6 cells in vitro was detected by Transwell migration and invasion assay.6.Western Blot technique was used to detect the phosphorylation of IL-8receptor CXCR2 and cytoskeleton-associated protein adhesion protein(FAK)Rac1/Cdc42 in MCF7/CLDN6 cells.Result:1.RT-PCR and Western-Blot techniques showed that the expression of CLDN6 in MCF7 / CLDN6 cells was significantly higher than that in MCF7 / Vector cells.2.RT-PCR technique showed that the expression of IL-8 in MCF7 / CLDN6 cells was significantly down-regulated compared with that in MCF7 / CLDN6 cells.3.Scratch test showed that the optimal concentration of IL-8 was 80 ng / ml and the best time was 48 h.The MCF7/CLDN6 cells were treated with the best concentration of IL-8 for 48 hours.4.Microscopic observation showed that the cells in IL-8 treated group were spindle-shaped mesenchymal cells.5.Compared with the control group,the fluorescence intensity of F-actin in the pseudopodia and the junction of the MCF7/CLDN6 cells was densely distributed and arranged regularly,compared with that in the control group.The results of biological electron microscopy showed that MCF7/CLDN6 cytoskeleton microfilaments arranged regularly,and the microtubule structure appeared and was located below the cell membrane.6.Transwell migration and invasion assay showed that the migration and invasion ability of MCF7 / CLDN6 cells was significantly increased compared with the control group.7.Western-Blot technique showed that the phosphorylation of IL-8 receptor CXCR2 and the cytoskeleton-associated protein FAKA Rac1 / Cdc42 in MCF7 /CLDN6 cells were up-regulated.Conclusion:CLDN6 regulates cytoskeleton rearrangement through down-regulation of IL-8/CXCR2 signaling pathway to inhibit migration and invasion of breast cancer MCF-7 cells.