Mechanism Of Bone Sialoprotein Through Integrin α_vβ₃Affecting ILK Signal Pathway In Human Breast Cancer Cells

Breast cancer is one of the top of female mortality malignant tumor，Metastasis is thekey cause of lose their life of the patients with breast cancer. Skeletal is one of the mostcommon target organs for breast cancer. It is very important for direct clinical therapy anddetermine therapeutic occasion and program that illuminating the molecular mechanism andsignaling transduction pathway related to tumor growth，invaison，metastasis will providenot noly a new targeting molecules and pathway of reversion of breast cancer, also findingnew tumor markers and ideal anticancer targeting drugs.Bone sialoprotein（BSP）is highly glycosylated，phosphorylated and sulfatedmultifunctional noncollagenous protein, the member of the Small Integrin-Binding LigandN-linked Glycoprotein（SIBLING）family of proteins that is essentially unique tomineralizing connective tissues，also expressed in a variety of malignant tumors. BSP playan important role in tumor cell adhesion，proliferation，invasion，matrix degradation，immune functions（inflammation and complement evasion），angiogenesis and metastasisthrough interactions with heterodimeric cell-surface integrins（primarily αVβ3）. Althoughmuch is known about mechanisms and signaling pathways by which other SIBLINGproteins（specifically OPN）regulate cancer progression，little is known about themechanisms and signaling pathways involved in BSP-mediated metastasis and tumorsurvival.Integrins as a membrane receptor family are a large class of adhesion molecules,Mainly mediated by cells with the extracellular matrix and cell-cell adhesion which wasfirst proposed by Hynes in1987. Intergin family of adhesion molecules are connected byalpha, beta subunit by non-colvalent bond connection from the heteodimeric transmembraneglycoprotein. Intergrin in tumor mainly act two aspects of the development:one mediated bytumor cells and extracellular matrix adhesion which can change the composition of thematrix,regulate the function of matrix biology,thereby contributing to tumor cell infiltrationand transfer. The other mediated by information from the extracellular matrix to theintracellular delivery.integrin α_vβ₃composed of αvsubunit and β3subunits is an importantmember of the integrin su family,part of the β3subunit group.the molecular connect the extracellular domain of the alpha, beta heterodimer into a spherical region, the intergincontains a divalent cation binding domain,through this combination of domain-specificrecognition of ligand arginine-glycine-aspartic acid（RGD） tripeptide sequence. α_vβ₃expression and proliferation of vascular endothelial cells is mediated by endothelial cellsand surrounding tissue binding,endothelial cells and tumor cell adhesion. In recent years,studies have shown that α_vβ₃plays an important role in tumor development,invasion andmetastasis. The present study found α_vβ₃ligand binding to promote FAK phosphorylationand cell transfer,play an important role in FAK signaling pathway in breast cancermetastasis.Integrin-linked kinase（ILK）is a59KD intracellular signaling protein,a Ser/Thrprotein kinase activity.In vivo,ILK excessive expression can promote proliferation andtumor formation:ILK can be dependent on PI3-K integrin and growth factor activation,andthus activated by phosphorylation of PKB,phosphorylated GSK-3inhibition,inhibition ofapoptosis promote cell proliferation.the protein kinase B（AKT）kinase pathway signals goto the main role of the ILK downstream traget proteins,the core of ILK/AKT signaltransduction pathway.activated AKT relocated to other parts of the cytoplasmic,nuclear orcell within the further phosphorylation of activation or inhibition of its downstreamsubstrates,influence tumor cell resistance to apoptosis,proliferation,migration and invasionand so on.The latest Gordon studies have shown that increased BSP expression directlystimulates the activation of the FAK-ERK signaling pathways,Phosphorylation of thetranscription factor c-Fos,the activation of AP-1,enhanced expression of MMP-2,MMP-9and MMP-14expression and cancer cells growth and migration.But which integrin is notinvolved. Our previous study the stability of siRNA recombinant retrovirus infection ofbreast cancer cell lines,231BO-BSP27cells which BSP gene stable silencing in breastcancer cells,while building a recombinant vector construction of pIRES2-hBSP-EGFP and aempty vector pIRES2-EGFP. In order to further explore the role of the BSP on regulation ofintracellular signal transduction pathway,the experimentts focus on this basis to theBSPgene silencing in breast cancer cells（referred231BO-BSP27）and import therecombinant vector construction of pIRES2-hBSP-EGFP was to restore the breast of BSP expression cancer cells（referred BSP27-hBSP）for the study,by western blotting and flowcytometry and immune cells chemical technology,research BSP gene silencing in breastcancer MDA-MB-231cells intgrated prime α_vβ₃expression levels and the restoration of theBSP expression after breast cancer231BO-BSP27cell integration elements α_vβ₃expressionlevel changes;MTT techniques and cell scratch assay integration factors of α_vβ₃specificinhibitor LM609biological functions;by western blotting was detected integrinα_vβ₃-specific inhibitor LM609breast cancer MDA-MB-231cells in the downstreamsignaling pathway of ILK/AKT signaling pathway.Depth the play of BSP expression andfunction of integrin α_vβ₃in brest cancer cells. In this study,the main findings are as follows:（1）.Not silence the control group and BSP gene231BO cells compared to the BSP genesilencing231BO-BSP27cells,BSP relative expression was significantly reduced（70.32±2.18）%（P＜0.01）,integrin α_vβ₃protein relative expression was significantly reduced（74.32±1.48）%（P＜0.01）;import recombinant vector construction of pIRES2-hBSP-EGFP wasto restore231BO-BSP27cell expression levels of BSP,the relative expression level ofintegration of prime α_vβ₃protein has been restored and restore（40.6±1.58）%（2）.Exogenous integration elements α_vβ₃,a specific inhibitor LM609deal with the BSPgene silencing group231BO-BSP27cells,and control group231BO cells,exogenous dilutethe concentration so that for10ug/ml the LM609inhibitors231BO,and231BO-BSP27cellsproliferative capacity decreased（44.50±0.92）%（50.32±1.32）%（P＜0.01）;10ug/mlLM609inhibitors to make231BO,231BO-BSP27cell migration significantly inhibited.（3）.Compared with the control group231BO cells,the BSP gene silencing group231BO-BSP27the relative expression levels of AKT and ILK in the cellsdecreased（;40.53±1.42ï¼›P＜0.01） and （33.78±1.51;P＜0.01）,exogenous dilution of10ug/ml of a specific inhibitor of LM609deal with the BSP gene silencing group231BO-BSP27cells and control group231BO cells,AKT and ILK relative expression levelscompared with before treatment,the relative expression levels were decreased （39.38±1.38ï¼›49.32±1.05; P＜0.01）...