The Study On The Mechanism Of RhBMP-2 Promoting The Stemness And Migration Of Breast Cancer Cells MCF-7

This study mainly discovered the molecular mechanism of rhBMP-2 promoting the stemness and migration of breast cancer cell line MCF-7.Firstly,ELISA assay was used to determine the endogenous expression of BMP-2 in MCF-7 cell line.Using cell lysis buffer to extract the total protein in the MCF-7 cell line,the expression level of BMP-2 was 356.46 pg/mg.As BMP-2 with heparin domain,in order to release and obtain the extracellular matrix,NaCl high salt buffer was used to extract the total protein.By using this method,the expression of BMP-2 was 499.49 pg/mg in MCF-7 cells.In addition,MCF-7 cells were induced with rhBMP-2 that BMP signaling pathway could be completely activated.The protein expression of CD44 was identified by Western Blot assay in MCF-7 cells,CD44 expression was up-regulated after rhBMP-2 induction.MCF-7 cells were transiently transfected with Smad4 siRNA and induced with rhBMP-2,the result indicated that up-regulation of CD44 in the process of promoting MCF-7 cell migration was through Smad signaling pathway.The protein expression of Rb was identified by Western Blot assay,Rb expression was down-regulated after rhBMP-2 induction.MCF-7 cells were transiently transfected with Smad4 siRNA and induced with rhBMP-2,the result indicated that down-regulation of Rb in the process of promoting MCF-7 cell migration was not through Smad signaling pathway.In order to further study which signaling pathway was involved in the process of migration,PI3 K and AKT inhibitors were used to block PI3 K and AKT signaling pathways respectively.It showed that PI3K-AKT signaling pathway played a role in the down-regulation of Rb in the process of promoting MCF-7 cell migration.Breast cancer stem cells could promote invasion and metastasis in breast cancer and CD44+/CD24-was the surface marker of breast cancer stem cells,whether the expression of cancer stem cells was increased in the process of rhBMP-2 promoting the migration of MCF-7 was unknown.The result of flow cytometry showed that the numbers of CD44+/CD24-group cells in MCF-7 cells were increased after rhBMP-2 induction.By using three-dimensional cell culture experiments,it showed that rhBMP-2 promoted the tumor spheroid formation in MCF-7 cells,and the proliferation rate of MCF-7 cells was reduced after rhBMP-2 induction.In the process of rhBMP-2 promoting MCF-7 cell migration,the expression of breast cancer stem cells and the stemness were increased.Besides,to detect the relationship of BMP-2,CD44 and Rb,40 cases of breast tumor specimens were tested using immunohistochemical experiment and statistical analysis.It showed that the expression of BMP-2 was mainly positively correlated with the expression of CD44,and negatively correlated with the expression of Rb.The promotion of migration induced by BMP-2 in breast tumors in vivo may be associated with CD44 and Rb.In summary,this study revealed that the rhBMP-2 promoted the migration of MCF-7 cells in vitro was related to the down-regulation expression of Rb and up-regulation expression of CD44.This study reveals the signaling pathways of rhBMP-2 promoting the migration of breast cancer cell line MCF-7 whith were relatived to the up-regulation of CD44 and down-regulation of Rb.In the process,the stemness of breast cancer stem cells was increased.