Identification Of Binding-related Protein Of BRBP1 Which Targets To Human Brain Metastatic Breast Cancer Cells

Breast cancer seriously threaten women’s health and lives.Breast cancer metastasize to bone,brain and lung increases the difficulty of breast cancer treatment and reduces the quality of life of patients.The median survival time of patients with brain metastases is short,with about80% mortality within one year of diagnosis,even small lesions can cause neurological disability.Study the mechanism of brain metastases in breast cancer,finding the target protein associated with brain metastasis on the membrane surface of breast cancer cells have great significance in the earlier diagnosis and treatment of brain metastases of breast cancer.A novel peptide,BRBP1(Patent number: ZL201210538671.4)was identified using an in vitro selection of a phage-displayed peptide library against human brain-seeking breast carcinoma cells(231-BR cells)previously in our lab.In vivo and in vitro studies showed that BRBP1 preferentially bound to the 231-BR cells.Not only that,BRBP1 peptide was able to specifically bind to human breast cancer axillary lymph node tissues ex vivo.Thus identification of the target protein of the peptide BRBP1 is of great importance to explore the mechanism of breast cancer brain metastasis、understand the value of BRBP1 peptide、broaden the role of BRBP1 peptide、strengthen the targeting of BRBP1 peptide.The BRBP1-Fc peptibody was eukaryotic expression then combined with Protein G magnetic beads,the proteins that could bind to the BRBP1-Fc in the total protein of 231-BR cells were obtained by Co-immunoprecipitation.At the same time,BRBP1-BIOTIN peptide interacted with streptavidin magnetic beads,then the proteins that bound to BRBP1-BIOTIN peptide in 231-BR cells membrane protein were pulled down.By silver staining and mass spectrum,14 proteins were obtained by BRBP1-Fc peptibody and 10 proteins were obtained by BRBP1-BIOTIN peptide.Our study further analyzed and identified whether these candidate proteins specifically bind to BRBP1.Objective: To detect the proteins’ expression of MVP,ERLIN2,RDX,AP2B1 and SND1 in human breast cancer cell lines 231-BR,MDA-MB-231 and MCF-7,and to identify whether these suspicious proteins interacts with BRBP1 peptide.Methods: 1.The mRNA levels of MVP,ERLIN2,RDX,AP2B1 and SND1 in human breast cancer cells 231-BR,MDA-MB-231 and MCF-7 were detected by real-time quantitative PCR.2.The expression of MVP,ERLIN2,RDX and AP2B1 proteins in human breast cancer cell lines 231-BR,MDA-MB-231 and MCF-7 was detected by Western blot,immunofluorescence and flow cyt.3.Design siRNA sequences,then transfection siRNA into 231-BR Cells to interfering protein expression.4.Verify the binding of 231-BR cells to BRBP1 peptide by Flow Cyt.Results: 1.The mRNA and protein’s expression of MVP was highly expressed in 231-BR、expressed in MDA-MB-231 and lowly expressed in MCF-7 cells.After interfering the expression of MVP,the binding rate of BRBP1 peptide to 231-BR cells increased.2.The mRNA of ERLIN2 was highly expressed in 231-BR and lowly expressed in MDA-MB-231 and MCF-7cells,the membrane protein’s expression of ERLIN2 was expressed in 231-BR and lowly expressed in MDA-MB-231 and MCF-7 cells.After interfering the expression of ERLIN2,no significant change in the binding of BRBP1 peptide to 231-BR cells.3.The mRNA of RDX was highly expressed in 231-BR cells,expressed in MDA-MB-231 cells,and lowly expressed in MCF-7 cells,but western blot,immunofluorescence and flow cyt results showed that the expression of RDX was higher in MCF-7 cells than in 231-BR and MDA-MB-231 cells.4.The mRNA of AP2B1 was highly expressed in 231-BR cells,expressed in MDA-MB-231 cells,lowly expressed in MCF-7 cells,but there was no significant difference in protein levels among the three cells.After interfering the expression of AP2B1,no significant change in the binding of BRBP1 peptide to 231-BR cells.5.There was no significant difference in the expression of SND1’s mRNA between 231-BR,MDA-MB-231 and MCF-7 cells.After interfering the expression of SND1,the binding of BRBP1 peptide to 231-BR cells was decreased slightly.Conclusion: After interfering the expression of ERLIN2 and AP2B1,the binding of BRBP1 peptide to 231-BR cells did not change;the expression of RDX on the surface of 231-BR cells is inconsistent with the expression of BRBP1 target protein.Therefore,ERLIN2,AP2B1 and RDX are not the target proteins that can bind to BRBP1 peptide.After interfering the expression of MVP,the binding of BRBP1 peptide to 231-BR cells was increased,the relationship of MVP and BRBP1 needs further verification.After interfering the expression of SND1,the binding of BRBP1 peptide to 231-BR Cells was decreased slightly,the relationship between SND1 and BRBP1 needs further validation.