| BackgroundBreast cancer is one of the most common malignancies in women.The incidence of breast cancer increased significantly in recent 10 years.Although the early detection of breast cancer increased significantly with the progress of medical technology and the mortality of breast cancer in women declined,half of those with breast cancer died of Breast cancer.In the Chinese population,the incidence of breast cancer in women with malignant tumors remains high.Breast cancer is not only a local tumor of the breast,but also a systemic disease.Nearly half of patients will still have recurrence and metastasis after surgery.Systemic transfer is an important cause of tumor recurrence and death.Therefore,the transfer of breast cancer is the leading cause of death in breast cancer,but also to study the treatment of breast cancer is a crucial node.However,there are still a large number of patients who are later found to have benefited from the development of modified surgery.Some patients benefit from traditional radiotherapy and chemotherapy.However,the traditional treatment methods have poor anti-tumor effect and lack of specificity.Toxicity and other shortcomings.Therefore,the discovery of new targets,the development of new anti-cancer drugs is essential.At present,molecular targeted therapy has made great breakthroughs and many molecularly targeted drugs have been used clinically.At present,epidermal growth factor receptor(EGFR)and vascular endothelial growth factor factor,VEGF)as a target,such as bevacizumab and so on,confirmed in clinical studies have a certain effect of clinical treatment.It has been reported that Neuropilin-1(abbreviation: NRP-1),a member of the Neuropilins family(NRPs),acts as a receptor for angiogenesis,development and tumor development of Semaphorin,VEGF and other factor co-The role of Neuropilin-2(abbreviation: NRP-2)is a member of the NRPs family and has 44% homology with NRP-1.Studies have found that NRP2 receptors are widely expressed in tumor cells and play an important role in lymphangiogenesis and tumor migration,invasion,proliferation,apoptosis and adhesion.In-depth study of the role of NRP-2 receptor in tumor biological behavior and its mechanism,to find a new。MethodsIn our laboratory,NRP-2 monoclonal antibody(NRP-2 m Ab)with high purity and concentration has been successfully prepared.The m RNA and protein levels of NRP-2 in different breast cancer cell lines were detected by RT-PCR and Western blotting.One of the cells with higher expression was screened for later experiments.Immunoflurescence assay was used to detect the expression of NRP-2 m Ab combined with NRP-2 activity and its specificity.The effect of NRP-2m Ab on the proliferation and proliferation of NRP-2m Ab breast cancer cell lines was examined by CCK8 assay.The morphological changes of breast cancer cells were observed under light microscope at different time points and drug concentration.-2m Ab on gastric cancer cell migration and invasion.The apoptosis of NRP-2 m Ab-induced cells was detected by flow cytometry.The adhesion of anti-NRP-2 monoclonal antibody to the cells with high NR-2 expression was studied by adhesion experiments.In breast cancer cells adhered to fibronectin(FN)experiments,rhodamine-phalloidin staining was used to study the effect of anti-NRP-2 antibody on the formation of stress cells in adherent cells.Confocal immunofluorescence microscopy was used to observe the effect of NRP-2 Co-localization with p-FAK,using common co-immunoprecipitation method to understand the relationship between NRP-2m Ab and α5β3.After the effect of NRP-2 m Ab was detected by Western blotting,the molecular changes of related signal pathways were observed.ResultsThe results showed that the expression of NRP-2 antigen on MDA-MB-231,MCF-7,BT-549 and SK-BR-3 breast cancer cell lines was expressed to varying degrees,and the localization was mainly on the cell membrane,MDA-MB-231 The m RNA and protein levels of NRP-2 were higher,and NRP-2 m Ab at different concentrations inhibited the proliferation,cell migration,invasion and adhesion of MDA-MB-231 breast cancer cell lines,and promoted its apoptosis.And NRP-2m Ab inhibited the growth and proliferation of MDA-MB-231 breast cancer cells in a concentration-dependent manner.Through confocal immunofluorescence detection by adhesion assay,it was found that NRP-2 m Ab can inhibit the formation of stress fibers in breast cancer cells,impede the spontaneous phosphorylation of tyrosine at FAK397 site,and thus interfere with the adhesion of breast cancer cells.Western blotting and common co-immunoprecipitation experiments showed that NRP-2 m Ab blocked the formation of complexes between NRP-2 and integrin α5β3,and decreased the expression of p-FAK,p-smad2/3,MMP-9,and Bcl-2 proteins.Expression of the Bax molecule.Conclusions1,NRP-2m Ab can specifically bind to NRP-2 protein on breast cancer cell membrane,and high expression of NRP-2 on MDA-MB-231 cells.2.NRP-2 m Ab significantly inhibited the growth of MDA-MB-231 breast cancer cells in a dose-dependent manner.3,NRP-2m Ab can significantly inhibit the migration and invasion of MDA-MB-231 breast cancer cells.4,NRP-2m Ab can significantly promote the apoptosis of MDA-MB-231 breast cancer cells.5,NRP-2m Ab can significantly inhibit the formation of stress fibers in MDA-MB-231 breast cancer cells,thereby affecting its adhesion.6.NRP-2 m Ab affects the phosphorylation of FAK by inhibiting the binding of NRP-2 and integrin α5β3.This discovery helps to deepen the understanding of the function of NRP-2 in cell adhesion and metastasis.NRP-2 mAb may directly or indirectly interfere with the growth of breast cancer cells by inhibiting NRP-2 and its receptor TGF-β1 to down-regulate the phosphorylation levels of downstream signaling pathways.8.NRP-2 m Ab inhibits NRP-2 related molecules,down-regulates the expression of MMP-9,Bcl-2 and other molecules,and up-regulates Bax apoptosis molecules.It can be found that NRP-2 interacts with NRP-2 and promotes migration,invasion and apoptosis of cancer cells.Hit and other related molecules. |
PDF Full Text Request