[Objective]To explore the role and mechanism of receptor activator of nuclear factor-κB ligand(RANKL)in bone metasta-sis of breast cancer.[Methods]The MDA-MB-231,BT549 and MCF-7 breast cancer cells were cultured in vitro,with the final concentra-tion of 200ng/ml RANKL(RANKL group),or the same volume of double distilled water(control,Ctrl group).Transwell,single-cell calcium imaging(SCCI),PCR,and western blot assays were performed.The bone marrow stem cells(BMSCs)of mice were isolated,and cultured with the supernatant of breast cancer cells,which contained RANKL.TRAP,ALP and von Kossa staining were performed.[Results]The relative migration cell numbers of RANKL added MDA-MB-231 cells[(1.7±0.1)vs(1.0±0.1),P=0.002]and BT549 cells[(1.6±0.1)vs(1.0±0.1),P=0.027]were significantly higher than those in the Ctrl group.The RANKL-200 group showed significantly higher levels of MCF-7[positive/total(％),58/127(45.7)vs 3/118(2.5),P＜0.001]and BT549 cells[positive/total(％),51/224(22.8)vs 31/228(13.6),P=0.011l]compared to the Ctrl group.PCR detection showed that E-cadherin in MDA-MB-231 cells of the RANKL-200 group was significantly lower than that in the Ctrl group,while the expression levels of N-cadherin and vimentin in the former were significantly higher than those in the latter.West-ern blot showed that the relative expression levels of N-cadherin were in the following order:RANKL-500 group＞RANKL-200 group＞Ctrl group(P＜0.05),whereas the expression levels of E-cadherin protein were in the order of RANKL-500 group＜RANKL-200 group＜Ctrl group.In the in vitro experiment of mouse bone marrow cells,the number of TRAP stained positive cells in the group containing RANKL su-pernatant was significantly higher than that in the Ctrl group[(451.3±15.0)vs(174.3±9.2),P＜0.001],while there was no statistically signifi-cant difference between ALP staining and Von Kossa staining(P＞0.05).[Conclusion]RANKL induced calcium signaling is mediated by NF-κB pathway,enhances the EMT process of breast cancer cells and promotes bone metastasis of breast cancer.
RANKLbone metastasis of breast cancercalcium oscillationEMTNF-κB signaling pathway
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