CLDN6 Inhibits Migration And Invasion In Breast Cancer Cell MCF-7 Via JAK2/STAT3 Signaling Pathway

Breast cancer is one of the most common cancer occurred in female.Metastasis is the main cause of death in breast cancer patients.Dysregulation of CLDNs is associated in tumorigenesis,progression and invasion in various types of cancer.Aberrant expression of CLDNs is involved in decreased or disordered tight junction(TJ)formation,which is one of the mechanisms involved in abnormal cell growth and tumorigenesis.In addition,CLDNs interacts with various signaling transduction pathways,allowing CLDNs directly participating in the control of gene expression.We discovered that over-expression of CLDN6 in breast cancer cell line MCF-7 suppressed cell migration and invasion in our previous work.Microarray showed that the transcription of JAKs/STATs signaling pathway genes,such as JAK2,STAT3,enhanced in MCF-7 cells over-expressing CLDN6,indicating that CLDN6 may inhibit migration and invasion in MCF-7 cell line by directly or indirectly interacting with JAK2/STAT3 signaling pathway.JAKs/STATs signaling pathway is the main downstream signaling pathway of cytokine,chemokine and growth factor receptor,which is involved in cell growth,proliferation,migration,invasion and other biological processes.However,the mechanisms in detail involved in CLDN6 inhibiting migration and invasion in MCF-7 cell line by via JAK2/STAT3 signaling pathway is not clear yet.Purpose:To demonstrate and discuss the mechanism that CLDN6 regulates migration and invasion in breast cancer cell line MCF-7 via JAK2/STAT3 signaling pathway,providing new therapeutic targets in breast cancer.Methods:1.To demonstrate that CLDN6 was over-expressed in MCF-7 cells,RT-PCR and western-blot was used.2.To detect the expression and activation of JAK2/STAT3 signaling pathway proteins,western-blot was used.3.To determine the maximum safe dose of JAK2 inhibitor AG490 to MCF-7 cells over-expressing CLDN6,RTCA iCelligence Analyzer was applied.4.To determine the optimum condition for AG490 to inhibit the phosphorylation of JAK2/STAT3 in MCF-7 cells over-expressing CLDN6,western-blot was used.5.To determine the effect of AG490 on migration and invasion of MCF-7 cells over-expressing CLDN6,wound healing assay and transwell assay were applied respectively.6.To determine whether EMT was involved in the inhibition of migration and invasion caused by CLDN6 via JAK2/STAT3 signaling pathway,western-blot was used.Results:1.CLDN6 was significantly over-expressed in Clone cells compared with Vector cells(P<0.01).2.JAK2/STAT3 signaling pathway was significantly activated in MCF-7 cells over-expressing CLDN6(P<0.05).3.IC50 of AG490 to MCF-7 cells over-expressing CLDN6 was 189?M.4.The optimum condition for AG490 to inhibit the phosphorylation of JAK2/STAT3 in MCF-7 cells over-expressing CLDN6 was 50?M(P<0.01)in concentration and 24h(P<0.01)in duration.5.AG490 treated MCF-7 cells with CLDN6 over-expressing showed significant enhancement in migration(P<0.05)and invasion(P<0.05).6.EMT was significantly inhibited in CLDN6 over-expressing MCF-7 cells and was enhanced after AG490 treated.Conclusion:CLDN6 inhibits EMT,migration and invasion in breast cancer cell MCF-7 by activating JAK2/STAT3 signaling pathway...