Screening And Identification Of The Binding Target Molecular Of BRBP1 Which Targets To Human Brain Metastatic Breast Cancer Cells

Breast cancer seriously endangers women’s health and life safety.Once brain metastasis occurs,the patient’s quality of life and survival time will be significantly reduced.The molecular mechanism of breast cancer brain metastasis is still unclear.Screening breast cancer cell brain metastasis-related target molecules and exploring the molecular mechanism of breast cancer brain metastasis are of great significance for finding potential diagnostic and therapeutic targets for brain metastatic breast cancer.In the early stage of this laboratory,the phage display peptide library screening technology was used to obtain the BRBP1 peptide(patent number: ZL201210538671.4).In vivo experiments with tumor-bearing mouse models also proved the peptide specifically binds to 231-BR cells.The test results of clinical specimens show that the peptide binds to the primary tissue specimens of human breast invasive ductal carcinoma and binds more strongly to the tumor cells of metastases.The binding specificity of BRBP1 peptide to breast cancer cell lines and clinical specimens suggests that the peptide may bind high-metastatic breast cancer cells and brain metastasis-related molecules on the surface.Exploring the target molecule of breast cancer brain metastasis cell line targeting peptide BRBP1 on the cell surface can help to further understand the molecular mechanism of breast cancer brain metastasis and provide a new entry point for the diagnosis and treatment of brain metastatic breast cancer.In the early stage of our laboratory,the BRBP1-Fc was co-immunoprecipitated with 231-BR cell total protein(Fc and 231-BR cell total protein Co-IP were used as controls),and the biotinylated BRBP1 peptide was co-precipitated with 231-BR cell membrane protein(Biotinylated BRBP1 peptide and MDA-MB-231,MCF-7 cell membrane protein Co-IP as controls).After silver staining of the immunoprecipitation products,the differential bands were analyzed and identified by mass spectrometry,but no target molecules were obtained.Therefore,this study further screened and identified target molecules that bind to breast cancer brain metastasis cell line targeting peptide BRBP1 through different methods.1.Identification of the binding candidate target molecule of breast cancer brain metastasis cell line targeting peptide BRBP1Objective: The expression of MSN and EFNB1 in breast cancer brain metastasis cell line 231-BR is higher than that of parental cell line MDA-MB-231,and it is related to cell metastasis and invasion.It may be the breast cancer brain metastasis cell line targeting peptide BRBP1 Target molecule.In addition,in the early stage of the laboratory,the suspected target molecule SND1 was obtained through screening by immunoprecipitation technology.Therefore,this chapter will further identify whether the three molecules MSN、 EFNB1 and SND1 are the target molecules of BRBP1.Methods: Construct overexpression plasmids based on MSN and EFNB1 respectively and transfect into MCF-7 cells to overexpress MSN and EFNB1,transfected into MCF-7 cells to overexpress MSN and EFNB1;Transfected si RNA-SND1 into 231-BR cells interfered with SND1 expression;flow cytometry identified the binding of the above cells to BRBP1 peptide.Results: pc DNA3.1(-)-MSN and pc DNA3.1(-)-EFNB1 overexpression plasmids were successfully constructed and successfully overexpressed in MCF-7 cells,but MCF-7 cells that overexpressed MSN and EFNB1 showed no increase in binding with BRBP1 peptide.si RNA-SND1 successfully interfered with SND1 expression in 231-BR cells,but 231-BR cells with down-regulated SND1 expression did not reduce binding to BRBP1 peptide.Conclusion: MSN、EFNB1 and SND1 are not the target molecules of BRBP1 peptide,and new research methods need to be adopted to re-screen suspicious target molecules.2.Screening for the binding target molecule of breast cancer brain metastasis cell line targeting peptide BRBP1Objective: Try to shorten or cyclize BRBP1 polypeptide to improve the affinity,and based on the Co-IP experimental ideas,use biotinylated peptides BRBP1-biotin and BRBP1-Fc1 peptibody to screen and identify target molecules.The 231-BR cells with high BRBP1 peptide binding and the 231-BR cells with low BRBP1 peptide binding were sorted by flow cytometry.The transcriptome was sequenced to obtain the differential genes,and the target molecules were further analyzed.Methods: The online database was used to predict the key sites of BRBP1 peptide binding to target molecules,design cyclic peptides and truncated peptides,and identify the binding performance.Using BRBP1-biotin Pull-down 231-BR cells protein(biotinylated unrelated peptide GK-biotin as a control),and the captured product was further identified by Western blot: the primary antibody is BRBP1-biotin,and the secondary antibody is Streptavidin-HRP.Based on the molecular weight of the target molecule captured by BRBP1-biotin as prompted by the Western blot results,the band corresponding to the silver stain of the captured product is selected for mass spectrometry analysis.In addition,the combination of BRBP1-Fc1 peptibody and 231-BR cells was fixed using the chemical cross-linking reagent DTSSP.Using Fc1 as a control,the total protein of the cells was extracted and the peptibody(or Fc1)and its coupling product were purified using Protein G magnetic beads Perform silver staining,mass spectrometry analysis of silver staining specific bands.Using flow cytometry,two sets of 231-BR cells with the strongest fluorescent signal(the most binding BRBP1-TAMRA)and the weakest signal(the least binding BRBP1-TAMRA)were obtained,followed by transcriptome sequencing,combined with the transcriptome reported in the literature Sequencing results,analysis and screening of differential genes and identification.Results: The binding performance of the two truncated linear peptides to 231-BR cells was weaker than that of BRBP1 peptide,and C6 and C9 cyclic peptides did not bind to 231-BR cells.In the Pull-down experiment,the BRBP1-biotin captured product showed no difference bands.The peptibody prokaryotic expression vector was successfully modified and expressed to obtain high-purity BRBP1-Fc1,no specific band was detected in the lane of BRBP1-Fc1 and cross-linked product.Through analysis and screening of the transcriptome sequencing results,8 candidate target molecules were obtained.Conclusion: Truncated or circularized BRBP1 peptide did not increase affinity with 231-BR cells.The BRBP1-biotin was used to capture cellular proteins through the Pull down experiment,and the BRBP1-Fc1 peptibody cross-linked cells did not find the BRBP1 peptide target molecule.8 candidate molecules screened by transcriptome sequencing analysis are currently undergoing further identification.In the early stage of this laboratory,the myeloid leukemia cell line HL-60 was used as the original cell,and the drug-resistance cell line HL-60 R was obtained by intermittent induction of increasing drug concentration,and used the phage display peptide library screening technology to obtain the 5R5 peptide combined with HL-60 R.The peptide has good binding specificity with leukemia resistant cell lines and bone marrow smears of patients with myeloid leukemia,suggesting that the peptide has the potential for clinical application,and the target molecule bound by the peptide may be related to drug-resistance of leukemia cells.We decided to construct a 5R5-Fc peptibody,hoping to achieve 5R5 peptide coupling with the Ig G Fc domain and enhance its affinity with the target molecule at the same time.Thereby enhancing its application value in the diagnosis and treatment of leukemia,it also lays the experimental foundation for the exploration of 5R5 peptide target molecules.1.Preliminary identification of clinical significance of 5R5 peptideObjective: To collect bone marrow smears of patients with clinical myeloid leukemia,and to identify the specificity of 5R5 peptide binding to bone marrow smears to initially identify the clinical significance of the polypeptide.Methods: Synthesize fluorescent modified peptide 5R5-TAMRA and control GK-TAMRA,divide each bone marrow smear into two areas,respectively incubate 5R5-TAMRA and GK-TAMRA,and identify 5R5 peptide and bone marrow smear by immunofluorescence binding specificity.Results: The 5R5 peptide did not bind to the bone marrow smears of four normal persons;in the bone marrow smears of 16 patients with myeloid leukemia,the 5R5 peptide bound strongly to 5 of them,9 to bind positively,2 to not bind,and the rate of binding positive is 87.5%.Conclusion: The 5R5 peptide has a good binding specificity with bone marrow smears of patients with myeloid leukemia.Comprehensive experimental results of the early 5R5 peptide specifically binding to leukemia drug-resistant cell lines at the cellular level suggest that the 5R5 peptide has clinical potential.2.Construction,expression and identification of eukaryotic expression plasmid vector of 5R5-Fc1 bivalent peptibodyObjective: The Fc domain of immunoglobulin Ig G1 can mediate a stronger immune effect function and effectively kill cells.It is a good choice for the development of peptibody drugs.This section uses the fusion of Ig G1 Fc and 5R5 peptide to construct a bivalent 5R5-Fc1 peptibody expressed in eukaryote.Methods: Eco RI and Nco I restriction enzymes double digested eukaryotic expression plasmid p FUSE-h Ig G1-Fc to obtain linearized vector fragments,and synthesized 5R5 gene with Eco RI and Nco I restriction sites and Gly-GLy-Gly-Ser linker fragment,T4 ligase enzyme linked linearized vector fragment and gene fragment;Eukaryotic expression plasmid vector p FUSE-h Ig G1-Fc-5R5 was transfected into 293 T cells for expression.Western blot was used to detect whether the 5R5-Fc1 peptibody was expressed in the culture supernatant.Flow cytometry was used to identify the binding properties of the 5R5-Fc1 peptibody to HL-60 R cells.Results: The eukaryotic expression plasmid vector p FUSE-h Ig G1-Fc-5R5 was successfully constructed,and the 5R5-Fc1 peptibody was successfully expressed and secreted into the cell culture supernatant.The binding rate of 5R5-Fc1 to HL-60 R is equal to the binding rate of Fc1 to drug-resistant cell line HL-60 R.Conclusion: Because the drug-resistant cell line HL-60 R expresses FcγR on the cell surface,the affinity between Fc1 and FcγR is higher than the affinity between 5R5 peptide and HL-60 R cells,resulting in the 5R5-Fc1 peptibody to bind "upside down" to drug-resistance cell line through its Fc1 and cannot achieve 5R5 peptide-dependent specific binding.Subsequent selection uses Ig G4 Fc with low affinity to FcγR to construct a tetravalent peptibody,while reducing the affinity between Fc and FcγR on the cell surface,and improving the binding ability of the peptide to the cell,realized 5R5 peptide-dependent specific binding between 5R5-Fc peptibody and HL-60 R cells.3.Construction,expression and identification of prokaryotic expression plasmid vector of(5R5)2-Fc4 tetravalent peptibodyObjective: Ig G4 Fc with low affinity to FcγR is selected,and the linker length between the peptide and Fc is extended.Two peptides are connected in each chain of the peptibody to construct a tetravalent(5R5)2-Fc4 peptibody to realize the 5R5 peptide-dependent specific binding between the peptibody and HL-60 R cells.Methods: Prokaryotic expression plasmid p ET-16b-(5R5)2-Fc4 was constructed and transformed into prokaryotic expression strain BL21 for prokaryotic expression.Coomassie brilliant blue staining to identify whether the peptibody is expressed.Flow cytometry identified(5R5)2-Fc4 peptibody bound to HL-60 R cells.Results: The prokaryotic expression vector p ET-16b-(5R5)2-Fc4 was successfully constructed and expressed to obtain the tetravalent(5R5)2-Fc4 peptibody.The binding rate of(5R5)2-Fc4 peptibody to HL-60 R is equal to the binding rate of Fc4 to HL-60 R.Conclusion: The affinity of 5R5 peptide to its target molecule is still weaker than that of Fc4 and FcγR,resulting in(5R5)2-Fc4 peptibody still binds "upside down" to the drug-resistant cell line HL-60 R due to the interaction between Fc4 and FcγR,and cannot specifically bind to HL-60 R depending on the 5R5 peptide.In subsequent experiments,it may be considered to modify the 5R5 peptide to improve the affinity or to mutate the Fc domain of the peptibody to eliminate its ability to bind to FcγR,and construct a peptibody that depends on the 5R5 peptide and specifically binds to the drug-resistant cell line HL-60 R.