| Tight Junctions(TJs) is a multifunctional complex which locates at the extreme apical region of junction-associated complexes in epithelial and endothelial cells, they have important roles in maintaining cell polari ty, regulating paracellular permeability and mediating intracellular signal transduction.TJs are composed with CLDNs, occlaudin and junctional adhesion molecules(JAMs) three kinds of membrane protein and endochylema protein zonula occludens(ZO)-1,2,3, Cingulin and Par3. CLDN multigene family is composed of27members and has been considered the main tight junction-forming proteins.CLDNs is the essential component of TJs, playing an important role in mainta-ining cell adhesion, polarity and signal transduction. Recent studies have reported that in some epithelium cancer for instance breast cancer, the abnormal expression of some CLDNs can result in the structural deleption and functional disorder of TJs, play an important role in the process of obtaining the malignant phenotype such as metastasis. There are at least27members in CLDNs family, CLDN6locates at16p13.3, encoding a23kDa protein composed with219amino acids, there are four transmembrne regions and two long loops which is the potential target of antibody therapy and C terminal related to signal transduction.In our previous work about the brest cancer suppressor, we cloned and identified CLDN6gene, whose expression was downregulated in hu man and rat mammary cancer cell lines. CLDN6, the main molecule, may act as an tumor suppress gene in the process of suppressing of metastasis. We construct an euacryotic expression vector and transfect human breast cancer cell MCF-7and obtain stable clones after G418screening. The biology phenotype of the clones changed, overexpreesion CLDN6significantly inhibit the proliferation, migration and invasion. But the mechanism how CLDN6influnce the phenotype of MCF-7is not clear. It has been reported that some members of CLDNs influence the biological behaviours of some particular cancers by activating or inhibiting some signaling pathway. CLDN7inhibits cell migration and invasion through ERK/MAPK signaling pathway in human lung cancer cells; CLDN1plays a causal role in the acquisition of invasive capacity in human liver cells and that c-Abl-protein kinase Cδ (PKCδ) signaling is critical for the malignant progression induced by CLDN1. We presume that CLDN6as a tumor suppressor may play a role in malignant events in the breast cancer by modulating some signaling pathways. Therefore, in our study, we progress experimental whole genome-scale screening to unveil the potential target genes and signaling pathways of CLDN6. Explore the functional mechanism of Tyk2/Stats signal pathway in the process of suppressing the malignant phenotype.Methods:1. Three derivative CLDN6expressing clones were analyzed using the Illumina Human HT expression beadchip V4for expression arrays. Choose6gene randomly from the expression changed genes:c-jun, TP53INPI, MAPK1, PTGS2, CD24and DAPK2, RT-PCR analysis their expression to confirm the accuracy of microarray;2. Analysis the target genes and signal pathway of CLDN6in MCF-7screened by microarray with bioinformatics methods;3. Western Blot was used to detect the expression and phosphorylation of members of Tyk-2/Stats:Tyk-2, Stat-1, Stat-3and Stat-4;4. Western Blot was used to determine the best concentration and time inhibit the Tyk-2/Stats signal pathway treatment of AG490(the inhibitor of Tyk-2/Stats signal pathway);5. After treated cells with AG490(best concentration and time), MTT was used to detect the proliferation of CLDN6overexpressing clones; Wound healing method was used to detect the magration of CLDN6overexpressing clones; Transwell experiment was used to detect the invasion of CLDN6overexpressing clones. Results:1. Microarry results:76genes collectively changed in three CLDN6overexpression MCF-7cell clones,54up-regulated and22down-regulated; RT-PCR results showing the expreesion of6genes choosed randomly from changed genes are confirmed to the results of microarray;2. Bioinformatics analysis results:Tyk-2, a member of Jak family and three members of Stats family:Stat-1, Stat-3and Stat-4were activated in CLDN6overexpression MCF-7cell clones;3. Western Blot results:The phosphorylation levels of Tyk-2, Stat-1, Stat-3and Stat-4were increased in CLDN6overexpression MCF-7cell clones;4. The best concentration and time of AG490treatment inbibit the phosphory-lation of Stat-1, Stat-3and Stat-4are25μM and above16hours;5. Treatment with AG490, the proliferation and migration ability were increased in CLDN6overexpression MCF-7cell clones, but there was no significant change of the invasion ability. AG490treatment reverse the function of CLDN6on cell prolifer-ation and migration.Conclusion:Breast cancer suppessor CLDN6inhibit the malignant phenotype of breast cancer cell MCF-7through activating the downstream target genes, the activation of Tyk-2/Stats signal pathway play an important role in inhibiting the breast cancer. |
PDF Full Text Request