The Role Of Semaphorin 4D Derived From Breast Cancer Cells In Inhibiting Osteoblast Differentiation In Vitro

Background and Objectives:Breast cancer is the highest incidence tumor in the female. Early diagnosis and treatment is good for recovery, but patients’ survival and quality of life are seriously affected once metastasis. Bone, especially the sternum and spine is the most common metastatic sites of breast cancer. There is no effective treatment except palliative care or surgical treatment is given priority to improve symptoms after bone metastasis currently. After breast cancer metastasis to bone, it usually lead to bone related events, including fracture, nerve compression, hypercalcemia, bone pain etc, the survival time and quality of life are often seriously influenced. Breast cancer cell metastases to bone generally cause osteolytic lesions, but bone matrix can’t be dissolved by breast cancer cell directly. The normal bone remodeling balance, which is dependent on the regulation of osteogenesis mediated by osteoblast and osteolytic mediated by osteoclast, is broken in cancer bone metastases. Cancer cells secrete a variety of factors to affect physiological activity of the osteoclasts or osteoblast in the process of the regulation, such as RANKL, PThrP, TGF-?, IL etc, and cause imbalance in bone remodeling, but more specific regulatory mechanism is yet unclear.Semaphorin 4D(Sema 4D) is a kind of axon guidance growth factor, and recently has been found highly expressed in lung cancer, prostate cancer, breast cancer and other malignant tumors, and closely related to the progress of malignant tumor. It’s reported that mouse osteoclasts could secrete Sema 4D, ant the latter could regulate the differentiation and migration of osteoblast, meanwhile the osteoblast differentiation and migration ability are strengthens in Sema 4D-/- mouse. So it is suggested that Sema 4D also involves in bone metabolism regulation in addition to participate in the axon guidance and tumor progression etc. We put forward hypothesis that Sema 4D derived from bone metastases breast cancer would play an important role in tumor progression and bone lesions. The aim of this study is to explore the possible effects and mechanisms of Sema 4D on breast cancer itself and osteoblast differentiation in breast cancer bone metastasis through shRNA interference and Transwell experiment, which simulates the microenvironment of breast cancer bone metastases, in order to offer a new theoretical basis for the prevention and treatment of breast cancer bone metastases.Methods:1. The Sema 4D expression in MDA-MB-231 and MCF-7 breast cancer cell line were detected by real-time fluorescent quantitative PCR, Western blot and cell immunofluorescence method, in order to screen the Sema 4D higher expression cell lines;2 Sema 4D shRNA lentivirus were transfected into high expression breast cancer cell lines, and screened by puromycin. The interference efficiency was determined by real time fluorescence quantitative PCR, western blot, cell immunofluorescence method;3 The cell proliferation, migration and cell cycle change were detected by cell counting kit-8( CCK-8), Transwell migration experiment, wound healing assay and flow cytometry after Sema 4D down-expression in breast cancer MDA-MB-231;4 In order to explore the role of Sema 4D in breast cancer bone metastases, we simulated the microenvironment of breast cancer bone metastases by transwell coculture system. The influence of Sema 4D on osteoblast differentiation induced by breast cancer cells were determined by real time fluorescence quantitative PCR, alkaline phosphatase staining and Alizarin red staining before and after Sema 4D interference;5. To determine the possible mechanisms of Sema 4D regulation of breast cancer bone metastases, we detected the AKT, p-AKT expression levels of osteoblast precursor cells before and after Sema 4D interference in transwell coculture system through Western blot.Results:1. The mRNA and protein expression of Sema 4D were higher in high invasive breast cancer cell line MDA-MB-231 than in low invasive breast cancer cell line MCF-7;2. Sema 4D shRNA could effectively down-regulated Sema 4D mRNA and protein expression level in breast cancer MDA-MB-231 cells, and Sema 4D down-expression breast cancer cell lines were successfully established;3. Down-expression of Sema 4D by shRNA can significantly inhibit the proliferation of MDA-MB-231 cells, block the cell c bycle at G1 phase, and suppress cell migration ability;4. Breast cancer cells obviously inhibited osteoblast precursor cells differentiation in transwell coculture system under osteogenic medium, while silent Sema 4D expression in breast cancer cell lines could weakened the inhibitory effect of osteoblast differentiation;5. Breast cancer cells obviously inhibited AKT phosphorylation of osteoblast precursor cells in transwell coculture system under osteogenic medium, while silent Sema 4D expression in breast cancer cell lines obviously increased the levels of AKT phosphorylation.Conclusions:1. Sema 4D is higher expressed in high invasive breast cancer MDA-MB-231 cell line;2. Sema 4D sh RNA effectively inhibits the Sema 4D expression in breast cancer MDA-MB-231 cells;3. Down-expression of Sema 4D obviously inhibits the proliferation, migration and cell cycle of breast cancer cell, Sema 4D may play a key role in the development of breast cancer;4. Down-expression of Sema 4D obviously weakens the inhibition effect of breast cancer cells on the osteoblast differentiation, breast cancer cells inhibits the osteoblast differentiation at least partly through Sema 4D;5. Sema 4D derived from breast cancer cells inhibits osteoblast differentiation possibly through inhibiting the phosphorylation levels of AKT.