CCL20 Mediates RANK/RANKL-induced Epithelial-mesenchymal Transition In Endometrial Cancer Cells

Objective: To explore the effect of receptor activator of NF-? B(RANK)/receptor activator of NF-? B ligand(RANKL)on the metastasis and epithelial-mesenchymal transition(EMT)of human endometrial cancer cell lines;To further investigate the role of chemokine ligand 20(CCL20)in the RANK/RANKL-induced EMT in endometrial cancer cells.Methods: The over-expression plasmid targeting RANK(p IRES2-3FLAG-EGFP-RANK)was constructed in vitro,and the empty plasmid(p IRES2-3FLAG-EGFP-CON236)was established as blank control group.HEC-1A and Ishikawa cells were transfected with lipofectamine technique to up-regulate the levels of RANK expression.Then,both HEC-1A/IshikawaControl cells and HEC-1A/IshikawaRANK cells were treated by 1ug/ml RANKL.Real-time fluorescent quantitative PCR(q RT-PCR)and western blot assay were applied to detect the m RNA and protein levels of RANK.The influence of RANK/RANKL on endometrial cancer cell migration,invasion and proliferation were tested by wound healing assay,transwell assay and CCK-8 assay.q RT-PCR,western blot and immunofluorescence assays were applied to detect the m RNA and protein levels of EMT markers,such as E-cadherin,N-cadherin,Vimentin,Snail and Twist.The expression and secretion of CCL20 in the RANK/RANKL-induced EMT in endometrial cancer cells were measured by protein chip analysis and enzyme-linked immunosorbent assay(ELISA).In vivo,human endometrial cancer orthotopic murine model was established;then q RT-PCR and ELISA were performed to detect the expression and secretion level of CCL20.Moreover,immunohistochemistry was used to assess the expressions of RANK,RANKL,E-cadherin,N-cadherin and Vimentin in human endometrial cancer tissue specimens with different stage(I,II,III).Spearman's correlation coefficient test was applied to analyze the expression correlation between RANK and E-cadherin,N-cadherin,Vimentin.Results: 1.RANK/RANKL expression was significantly elevated in endometrial cancer tissue of higher stage.Also,RANK level was positively connected with N-cadherin(p=0.0229)and Vimentin(p=0.0398),but negatively with E-cadherin(p=0.0118).2.The migration and invasion capabilities of HEC-1A and Ishikawa cells were significantly promoted by RANK/RANKL(P< 0.01).However,the proliferation ability was not changed.3.RANK/RANKL up-regulated the m RNA and protein levels of N-cadherin,Vimentin,Snail and Twist(P< 0.05)in HEC-1A and Ishikawa cells,but down-regulated the expression of E-cadherin(P< 0.05).4.The expression and secretion level of CCL20 were notably increased(P< 0.001) in RANKL-treated HEC-1ARANK and IshikawaRANK cells.5.RANK/RANKL elevates the expression and secretion level of CCL20(P< 0.01)in nude mice orthotopic transplantation modes.6.Both exogenous and endogenous CCL20 facilitates invasion of HEC-1ARANK cell.Moreover,the expression of E-cadherin was decreased,whereas N-cadherin,Vimentin and Twist were increased in CCL20-stimulated HEC-1ARANK cells.Conclusions: 1.RANK/RANKL could induce EMT in HEC-1A and Ishikawa cells,and promotes metastasis.2.RANK/RANKL up-regulated the expression and secretion level of CCL20 in endometrial cancer cells,which accelerated migration/invasion and promoted cancer progression through EMT.