The Construction Of Human B7H4 Promoter Luciferase Reporter Gene Vector And The Regulation Of Transcription Factor P53

Object:Tumor immune escape is the major obstacle to the successful clinical therapy,and higher expression of immunosuppressive molecules in the tumor microenvironment is the major mechanism of tumor immune escape.B7H4(B7 homology 4)is one of immunosuppressive molecules belonging to B7 family.Previous studies suggest that that higher expression of B7H4 in the tumor microenvironment is associated with the tumorigenesis,disease progression of most common malignant tumors and patients prognosis.However,the reasons for up-regulation of B7H4 expression in tumor cells or tumor infiltrating immune cells are still not clear.This project is aimed to construct human B7H4 promoter luciferase reporter vector and to explore the transcription factors or inducilbe factors in the tumor microenvironment which can induce B7H4 expression.This study will help to set up the platform for the further research on the mechanisms of B7H4 in the tumor immune escape.Methods:1.Screening and indentificaiton of human and murine breast cancer cell lines which expressed B7H4 protein using Western Blotting and flow cytometry.2.Analysis of inducible factors of B7H4 expression on the membrane surface of murine breast cancer cell.Spleen cells,CD4+ T cells and macrophages were isolated from C57BL/6 mouse,then activated by Con A,PMA/ionomycin and LPS respectively.After murine breast cancer cell E0771 were cocultured with these activated immune cells or inflammatory factors respectively,including LPS,IFN-g,TNF-a and IL-4,the expression of B7H4 were analyzed with flow cytometry(FACS).3.The construction of E0771 in situ tumor bearing mice model Murine breast cancer cell E0771 were subcutaneously injected in breast region around areola of C57BL/6 female mice,tumor infiltrating immune cells were isolated from tumor tissue and detected the expression of membrance B7H1 and B7H4 protein by FACS at 6,12,24 days after tumor cells injection.4.The construction of h B7H4-promoter luciferase reporter vector Three h B7H4 promoter sequences of different lengths were amplificated by PCR,then linked with the report gene vector p GL3-Basic and indentified by DNA sequencing.293 T cells were transfected with three human B7H4 promoter recombinant vectors and p RL-TK plasmid respectively,then detected the transcriptional activity by luciferease assay.5.Construction of human p53-CRISPR/cas9 n plasmid3 pairs of sg RNA sequence targetting on p53 gene exon were synthesized and linked with p X461 plasmid.293 T cells were transfected with recombinant p53-CRISPR/cas9 n plasmids and the efficiency of p53 knockout were analyzed using Western Blotting.7.Human breast cancer cell SK-BR3 were transfected with recombinant p53-CRISPR/cas9 n plasmids and the expression of p53 and B7H4 were detected by Western Blotting.Results:1.The expression of B7H4 in human breast cancer(MCF-7,MDA-MB-468,and SK-BR3)and murine breast cancer(4T1,E0771)can be detected by Western Blotting,but membrance B7H4 were only found on the the surface of breast tumor cells MDA-MB-468 and SK-BR3 by FACS.2.Up-regulation of B7H1 expression on the surface of murine breast cancer E0771 were detected by FACS after coculture with activated imunune cells(CD4+T)or inflamatory cytokines(IFN-g)and the expression of B7H1 were upregulated on the surface of tumor infiltrating immune cells isolated from in situ tumor bearing mice model on the sixth,twelfth,twenty-fourth day.however,there are no change for B7H4 protein.4.The construction of h B7H4 promoter luciferase reporter vectors is comfirmed by DNA sequencing.p GL3-h B7H4-0.5kb plasmid showed the highest transcriptional activity by luciferease assay,campared with p GL3-h B7H4-1kb and p GL3-h B7H4-2kb.6.The construction of human p53-CRISPR/cas9 n plasmid is comfirmed by DNA sequencing.The combination of p53-h B7H4-1T1/B1 or p53-h B7H4-1T2/B1 plasmids showed higher knockdown efficiency for p53 gene.Conclusion:No change of B7H4 expression on the surface of murine breast cancer cell E0771 in the tumor cell-immune cells coculture system or E0771 tumor bearing modle,indicating that the regulatory mechanisms of B7H4 expression may be different from that of B7H1.B7H4 may be constitutively expressed in tumor cell.The construction of h B7H4 promoter luciferase reporter vectors will provide the tools for the analysis of B7H4 prmoter binding sites for transcription factors.Preliminary result showed that B7H4 exprssion is inhibited by p53 gene knockdown using p53-CRISPR/cas9n system.