Siglec-15 Promotes The Proliferation And Migration Of Colorectal Cancer Cells And Inhibits The Infiltration Of CD4~+ And CD8~+T Cells In Tumor Tissues

Backgroud:Sialic acid-binding immunoglobulin-type lectin-15(Siglec-15),as one of the Siglec gene family members,could contribute to tumor immune escape by inhibiting T cell function and is identified as a novel immunosuppressive molecule,whose sequence is similar to PD-L1,but its immunosuppressive function is independent of PD-1/PD-L1.Siglec-15 is highly expressed in a variety of human tumor cells and tumor-associated macrophages,but its expression and function in colorectal cancer remain unclear.IFN-γcan activate the inflammatory signal transduction pathway JAK-STAT1/3.It has been reported that IFN-γcan down-regulate the expression of Siglec-15 in RAW264.7macrophages.However,there is no relevant report on the effect of IFN-γon Siglec-15expression in colorectal cancer(CRC)cells and its regulatory mechanism.In this paper,we will explore the expression and function of Siglec-15 in CRC and preliminarily analyze the molecular mechanism of IFN-γregulating the expression of Siglec-15 in CRC cells.Objective:To analyze the expression and biological function of Siglec-15 in CRC;To explore regulation mechanism of IFN-γon the expression of Siglec-15 in CRC cells.Methods:1.The effect of Siglec-15 on the function of CRC cells and immune cell infiltration in CRC microenvironmentThe m RNA expression level of Siglec-15 was assessed in human CRC tissues versus normal colon tissues from the TCGA m RNA data set.The protein level of Siglec-15 was determined via IHC staining analysis in a tissue microarray containing 52paired CRC and adjacent tissues.Moreover,Siglec-15 expression in CRC cells was detected by western blot.sh RNA knockdown of Siglec-15 plasmid was constructed and transfected into human colon cancer cells SW480.CCK8 assay and wound healing assay were used to determine the proliferation and the metastasis ability of SW480 cells.CD8~+T cells were isolated from mouse spleen with anti-CD8 magnetic beads,and were co-cultured with mouse CRC cells MC38.The cytotoxic effect of CD8~+T cells on MC38was estimated using CCK8 assays and cytotoxicity detection kit.ELISA was used to detect the secretion of cytokines TNF-αand IFN-γ.Mouse xenograft model was established by subcutaneous injection MC38 cells-overexpressed Siglec-15.The infiltration of CD4~+,CD8~+、Treg immune cells in xenograft tumors and human colorectal cancer tissues were detected by immunohistochemistry.2.The effect of IFN-γon the expression of Siglec-15 in CRC cells and its regulatory mechanismSW480 cells were treated with IFN-γ(0,25,50,100 ng/ml)for 48 hours.The expression level of Siglec-15 was detected by western blot.The binding sites of transcription factor STAT1,STAT3 on Siglec-15 promoter was predicted using JASPAR database.The effect of IFN-γon Siglec-15 was analyzed by STAT1 inhibitor Fludarabine,STAT3 inhibitor Stattic,or sh RNA knockdown of STAT1 and STAT3.Results:1.TCGA data analysis,immunohistochemical and Western blot results indicated that Siglec-15 was highly expressed in CRC.CCK8 assay and wound healing assay showed that Siglec-15 knockdown could inhibit the proliferation and migration of SW480 cells.Overexpression of Siglec-15 suppresses CD8~+T-cell-mediated killing of tumor cells and the secretion of IFN-γand TNF-αin vitro,and facilitates tumor growth in vivo.High levels of Siglec-15 in CRC tissues are associated with decreasing infiltration of CD4~+T and CD8~+T cells in tumor microenvironment.However,there was not statistical significance in the infiltration of Foxp3~+Treg cells.2.Western bolt data showed that Siglec-15 protein levels were decreased following IFN-γtreatment,especially at 100ng/ml.STAT1 inhibitor or STAT1 silencing blocked IFN-γinduced down-regulation of Siglec-15 protein levels in SW480 cells,while no significant effect was observed with STAT3 inhibitor or STAT3 silencing.Conclusion:Siglec-15 is highly expressed in CRC,and it may contribute to the progression of colorectal cancer by promoting the proliferation and migration of CRC cells and hampering CD4~+T cells and CD8~+T cells infiltration in the tumor microenvironment.IFN-γinduced downregulation of Siglec-15 in colorectal cancer cells may be attributed to transcription factor STAT1.