The Biological Role Of Semaphorin 4D In Human Breast Cancer Development And Research On Its Molecular Mechnism

Objective:Semaphorin 4D(Sema4D)is highly expressed in many human tumors and functions in the regulation of immune function,epithelial morphogenesis,angiogenesis,invasion and metastasis.However,the biological roles of Sema4D in breast cancer remain undefined.This study was designed to investigate the effects of Sema4D on proliferation,cell cycle progression,apoptosis,invasion,migration,tumor growth and explore molecular mechanism in breast cancer.Our research might provide a research base to explore the possibility of Sema4D as a potential biomarker and target for diagnosis and treatment of human breast cancer.Methods:Immunohistochemical analysis was applied to determine that levels of Sema4D in 40 cases of human breast cancer samples.The expression level of Sema4D was investigated in seven breast cancer cell lines,including MDA-MB-231 and MDA-MB-468 cells.Then,the MDA-MB-231 and MDA-MB-468 cells with Sema4D down-regulation were established by infection with a lentivirus encoding Sema4D-shRNA.To evaluate the effects of Sema4D on cell proliferation,cell cycle progression,apoptosis,invasion and migration,MDA-MB-231 and MDA-MB-468 cells with Sema4D down-regulation were analysed by MTT assay,flow cytometry,wound-healing assay and transwell experiments in vitro.To evaluate the effects of Sema4D on tumor growth,MDA-MB-468 cells with Sema4D down-regulation were injected in BALB/c nude mice subcutaneously,and then Sema4D expression level were evaluated by immunohistochemical analysis,meanwhile tumor volume was determined by caliper measurement and calculated by established methods.In order to explore the molecular mechanism of Sema4D on apoptosis and cell cycle,the cell cycle related proteins(p27,p21 and CylinD1)and the apoptosis related proteins such as Caspase3 and PARP were detected by Western Blotting.Down-stream signaling molecules of the Sema4D/Plexin-Bl were determined by Co-Immunoprecipitation and MALDI-TOF mass spectrometry(MS).The interaction between Plexin-B 1 and its down-steam signaling molecules were then confirmed by Western Blotting.Meanwhile,the levels of phospho-ERK and phospho-RhoA were detected by Western Blotting respectively.Results:Immunohistochemical analysis about 40 cases of human breast cancer revealed that high levels of Sema4D and 60%of breast cancer samples exhibited moderate and strong Sema4D expression.The level of Sema4D was highly related to the tumor size,TNM stage and the type of triple negative breast cancer.Sema4D were significantly expressed at higher levels in MDA-MB-231 and MDA-MB-468 cell lines compared with the normal human breast epithelial cell lines MCF10A and 184A1.The expression of Sema4D was significantly down-regulated in MDA-MB-231 and MDA-MB-468 cells after infection with a lentivirus coding for Sema4D-shRNA,compared with uninfected cells.MDA-MB-231 and MDA-MB-468 cell proliferation was significantly inhibited in the Sema4D-shRNA group,whereas the proportions of cells in the G0/G1 phase and undergoing apoptosis were significantly increased.In addition,the invasion and migration ability of these cells were obviously reduced.The growth of MDA-MB-468 xenotransplanted tumors with down-regulated Sema4D were inhibited comparing with control group.The levels of CyclinD1 were dramatically reduced in MDA-MB-231 and MDA-MB-468 cells with dowm-regulated Sema4D,while the levels of p27 and p21 were dramatically increased,which caused G1 phase arrest.Consistent with the result of cell apoptosis in flow cytometry,it turned out an activating of apoptosis related proteins such as Caspase3 and PARP.The down-stream signaling molecules of the Sema4D/Plexin-B 1 pathway were detected by Co-Immunoprecipitation and MALDI-TOF mass spectrometry(MS),the results showed that ERK and RhoA might be down-stream signaling molecules of Sema4D/Plexin-B1 axis.The levels of phospho-ERK and phospho-RhoA were both inhibited by down-regulation of Sema4D,which may probably cause the effects of Sema4D on proliferation,cell cycle progression,apoptosis,invasion and migration in breast cancer cells.Conclusion:60%of breast cancer samples exhibited moderate and strong Sema4D expression.Sema4D was expressed at significantly higher levels in MDA-MB-231 and MDA-MB-468 human breast cancer cell lines.Down-regulation of Sema4D expression by shRNA interference significantly inhibited the proliferation,invasion and migration of both MDA-MB-231 and MDA-MB-468 cells.Meanwhile,cell apoptosis and blockade of the cell cycle at the G1 phase occurred in these cell lines,respectively.The growth of MDA-MB-468 xenotransplanted tumors with down-regulated Sema4D were inhibited comparing with control group.The results showed that Sema4D/Plexin-B 1 could promote the biological role of breast cancer by activating incellular signaling pathway,including ERK and RhoA down-stream signaling molecules,which expanded the knowledge about the role of Sema4D/Plexin-B 1 in the development of breast cancer.Our results showed that Sema4D might represent a potential biomarker and target for treatment of human breast cancer.