The Role And Mechanism Of Active Vitamin D Mediated By Bmi-1 In Inhibiting The Allograft Growth And Bone Metastasis Of Breast Cancer

Background:Breast cancer is a common female malignancy and the second leading cause of death in women with malignant tumors.Although advances in the diagnosis and treatment of breast cancer have continued,they have not led to a significant increase in overall survival.The main reason is that breast cancer is prone to distant metastases.Clinical trials have shown that bone is the most common distant metastasis organ of breast cancer,and bone metastases occur in about 70%of patients with breast cancer during natural course.After the occurrence of bone metastases in breast cancer,due to the increased destruction of bone structure,skeletal related adverse events are prone to occur,which seriously affects the quality of life of patients.What is even more serious is that once breast cancer is transferred to bone,it cannot be cured.An ongoing clinical study shows that vitamin D deficiency is a common risk factor for bone metastases in breast cancer,and normal vitamin D levels may prevent bone metastases.However,the mechanism of active vitamin D deficiency in the development of breast cancer and bone metastasis has not been reported.Objectives:1)To clarify the role of active vitamin D in inhibiting breast cancer cell growth and bone metastasis;2)To clarify the role of active vitamin D mediated by Bmi-1 in inhibiting the allograft growth and bone metastasis of breast cancer;3)To reveal the mechanism of active vitamin D in inhibiting the occurrence and metastasis of breast cancer,and provides an experimental and theoretical basis for the clinical use of active vitamin D in the prevention and treatment of breast cancer and its bone metastases.Methods:A murine breast cancer TM40D cells were treated with exogenous different concentrations of 1,25?OH?₂D₃,the proliferation,migration and invasion of murine breast cancer TM40D cells were examined using CCK-8 assay,scratch test,and transwell assay,respectively.The murine breast cancer TM40D cells were transplanted into tibiae of WT or Cyp27b1^-/-female mice treated with or without exogenous 1,25?OH?₂D₃,and the phenotype of bone metastases was analyzed using imaging and histopathological methods.A murine breast cancer cell line TM40D stably transfected with down-regulating Bmi-1 gene was established.The effects of Bmi-1 knockdown on the proliferation,migration,and invasion of mouse breast cancer TM40D cells were examined using the CCK-8 assay,scratch test,and transwell assay.Murine breast cancer TM40D cells transfected with an empty vector or Bmi-1 knockdown TM40D cells were transplanted into the subcutaneous or upper tibial medullary cavity of WT or Cyp27b1^-/-mice,and then treated with or without exogenous 1,25?OH?₂D₃,the phenotypic changes of transplanted tumors and bone metastases were analyzed using imaging,histopathology and molecular biology methods.Results:1)1,25?OH?₂D₃ inhibited the proliferation,migration and invasion of breast cancer TM40D cells in vitro;2)1,25?OH?₂D₃ inhibited the tumor growth and osteolytic destruction of breast cancer TM40D cells in bone tissue;3)1,25?OH?₂D₃inhibited osteoclastic resorption of the bone induced by breast cancer cells;4)1,25?OH?₂D₃ can significantly down-regulating the expression of Bmi-1 in breast cancer cells in vivo and in vitro;5)Bmi-1 knockdown inhibited tumor cell proliferation and accelerated tumor cell senescence by elevating the expression levels of P16,P19,and P53,and inhibited the allograft growth of breast cancer;6)Bmi-1knockdown suppressed the proliferation,migration and invasion of breast cancer TM40D cells in vitro;7)Bmi-1 knockdown suppresses osteolytic destruction produced by breast cancer cells in vivo;8)Exogenous l,25?OH?₂D₃ and Bmi-1knockdown both up-regulated the expression of tumor suppressor genes in breast cancer cells in tibiae.Conclusions:The results of this study indicate that active vitamin D can inhibit the occurrence of breast cancer and bone metastasis by down-regulating Bmi-1,activating of P16/Rb and P19/P53 signaling pathways,inhibiting breast cancer cell proliferation and growth in bone tissue,inducing breast cancer cell senescence,and reducing osteolytic destruction.The results of our study not only reveal the mechanism of action of active vitamin D in inhibiting the occurrence and metastasis of breast cancer,but also provide experimental and theoretical basis for the clinical application of active vitamin D in the prevention and treatment of breast cancer and its bone metastases.