The Study Of MUC1 Integrates PD-L1 In Human Breast Cancer

Object: In the tumor microenvironment,there are certain immune negative regulatory mo-lecules,especially PD-L1 molecules,which can not only promote tumorigenesis,development,but also participate in tumor immune escape.As a transmembrane gl-ycoprotein,MUC1 is highly expressed in breast cancer tissues and participates in a variety of tumor immune responses.Recent studies have confirmed that the expression of PD-L1 molecules is downregulated after targeting MUC1 in NSCLC(non-small cell lung cancer).The purpose of this project is to study the regulation of PD-L1 expression by MUC1 in breast cancer,and to lay a certain experimental basis for the subsequent study on PD-L1 expression regulation.Methods: 1.Analysis the expression of PD-L1 and MUC1 in breast tissue.Use the pathological tissues,which contain breast cancer tissues and normal tiss-ues adjacent to cancer,whitch were collected from 14 patients with breast canc-eras experimental specimens,and randomly divided them into experimental gro-ups and control groups.Immunohistochemical staining was used to label PD-L1 and MUC1 molecules.Semi-quantitative calculation was performed based on the p roportion of positive molecular volume,and the expression of PD-L1 and MUC1 was counted to analyze the correlation between MUC1 and PD-L1 in breast cancer specimens.2.Screen PD-L1 and MUC1 positive expression human breast cancer cell lines.The expression of MUC1 and PD-L1 in human breast cancer cell lines(MX-1,SKBR3,MDA-MB231,MCF-7,HCC1937)was detected by Western Blot(WB)and flow cytometry(FCM).The cell lines expressing both PD-L1 and MU-C1 were screened for subsequent experiments.3.Overexpression of MUC1 recombinant vector and construction of targeting MUC1 recombinant lentiviral vector.The MUC1-m RNA sequence was amplificated by PCR then linked with the vector p EX-1 to construct p EX-1-MUC1 recombinant plasmid;sh RNA primers were designed according to MUC1-m RNA sequence,further annealing,ligation,lentivirus coating,and construct the recombinant lentivirus sh RNA-MUC1 that targeted MUC1.4.Preliminary validation MUC1 regulation the expression of PD-L1 in vitro.After the recombinate of p EX-1-MUC1 plasmid and sh RNA-MUC1 lentivirus.Breast cancer cell lines were infected with the recombinant plasmids and Lentivirus then detected the expression of PD-L1 by Qpcr,WB and FACS.5.The detection of preliminarily mechanism of MUC1 how to regulatory PD-L1 in breast cancer cells.The DNA sequence of the PD-L1 promoter region was ligated to the luciferase reporter vector p GL3-Basic to construct a dual-luciferase reporter system for the h uman PD-L1 promoter,which was then transfected with the breast cancer cells b efore and after treatment.The principle of the enzyme activity reporting system was used to test the strength of the promoter.After treating the cells with the NF-k B-p65 pathway inhibitor BAY-8075,the activity of the cell promoter was compared before and after treatment.Results: 1.Detective results show: MUC1 expression in breast cancer is higher than no-rmal tissues;Clinical breast cancer specimens of network stations show that the re are positive correlations of MUC1 and the expression of PD-L1.2.Successfully screened PD-L1 and MUC1-positive human breast cancer cell lines(MD-MBA231,MX-1,SKBR3);3.The MUC1 overexpression recombinant vector(p EX-1-MUC1)and the MUC1 targeting lentivirus vector(sh RNA-MUC1)were successfully constructed;4.The q PCR and WB results showed that after overexpression of MUC1 in br-east cell line MB231,the corresponding PD-L1 m RNA and protein levels were up-regulated compared to the control group;three breast cancer cell lines(MB231,MX-1,SKBR3)PD-L1 expression was down-regulated after targeted MUC1 treatment;5.In the double luciferase activity verification experiment,after overexpression of MUC1 in breast cell line MB231,the corresponding PD-L1 promoter activity was increased compared to the control group;three breast cancer cell lines(M B231,MX-1,SKBR3)After targeting MUC1,the activity of PD-L1 promoter was decreased to a certain extent compared with the control group;the promo-ter activity of three groups of breast cancer cells after BAY was lower than that of the control group.Treatment groups.Conclusion: In human breast cancer,MUC1 can regulate PD-L1 expression,and may influence the by PD-L1 promoter activity by NF-k B pathways,and thus play a role of PD-L1 expression regulation.