Experimental Study Of The Effect Of FUT8 On The Biological Function And Tumor-associated Macrophage Phenotype Of Triple-negative Breast Cancer Cell Lines

BACKGROUND Breast cancer is one of the most common tumours in women,particularly Triple-negative breast cancer(TNBC),characterised by estrogen receptor(ER),progesterone receptor(PR)and human epidermal growth factor receptor 2(HER-2)negativity,which is one of the most malignant subtypes of breast cancer.Blocking Programmed death ligand 1(PD-L1)have been clinically applied in other types of tumours,but the effectiveness in TNBC remains unsatisfactory,so there is a need to understand the regulatory mechanisms of PD-L1 in TNBC to better serve the diagnosis and treatment.Fucosylation is a glycosylation process in which fucose is transferred from GDP-fucose to other molecules by fucosyltransferases(FUTs).α1,6-fucosyltransferase(α-1,6-fucosyltransferase),a fucosylase,encoded by the FUT8 gene,which is involved in the biosynthesis of N-linked oligosaccharides in glycoproteins and influences tumour progression through modification of glycoproteins.It is an important role in the Epithelial-mesenchymal transition in hepatocellular carcinoma.In recent years,FUT8 has also been found to be strongly associated with poor prognosis in breast cancer,but the biological function of FUT8 in triple-negative breast cancer,particularly with regard to invasion and metastasis,is not clear.Tumor-associated macrophages is an important component of the tumor microenvironment and influence tumor progression by regulating various components within the tumor,but the role of FUT8 in altering the phenotype of tumor-associated macrophages in triple-negative breast cancer has not been reported.We will investigate the biological function of FUT8 in triple-negative breast cancer cell lines and its effect on tumour-associated macrophages.OBJECTIVE 1.To investigate the effect of FUT8 on the proliferation,cell cycle,migration,and invasive ability of two Triple-negative breast cancer cell lines,MDA-MB-231 and MDA-MB-468.2.To investigate the effect of FUT8 on PD-L1 mRNA as well as protein expression in Triple-negative breast cancer cell lines.3.To investigate the tumor-associated macrophage phenotype of FUT8 in the Triple-negative breast cancer microenvironment.MATERIALS AND METHODS 1.Using siRNA to interfere with FUT8 expression and thus investigate its effect on the biological functions of Triple-negative breast cancer cell lines(1)Cell Counting Kit-8(CCK-8)kit to detect the effect of FUT8 interference on the proliferative capacity of Triple-negative breast cancer cell lines.(2)Cells were stained with propidium iodide and the effect of FUT8 interference on the cell cycle of Triple-negative breast cancer cell lines was examined by means of flow cytometry.(3)Scratch assay to detect the effect of FUT8 interference on the migration ability of Triple-negative breast cancer cell lines.(4)The effect of FUT8 interference on the invasive ability of Triple-negative breast cancer cell lines was examined by Transwell matrix assay.2.Real-time quantitative fluorescent PCR and Western blot assays were performed to investigate the effect of FUT8 on PD-L1 expression in triplenegative breast cancer cell lines.3.A Triple-negative breast cancer-tumor-associated macrophage co-culture model was constructed to study the effect of FUT8 on macrophage phenotype.Statistical results were considered statistically different at P<0.05.RESULTS 1.The proliferation ability of both cell lines was reduced after FUT8 interference,and the cell cycle G0/G1 phase and S phase were shortened(P<0.05).2.The scratch assay showed that the motility migration ability of both cell lines was reduced after FUT8 interference,and the number of invading cells after FUT8 interference was smaller than that of the NC group in the Transwell stromal assay.Real-time quantitative fluorescence PCR and Western blot experiments were performed to detect the mRNA of CDH1,CDH2 and VIM and the encoded proteins in each treatment group.mRNA and protein levels were found to be increased in CDH1 and decreased in CDH2 and VIM after FUT8 interference(P<0.05).3.For both cell lines after FUT8 interference PD-The mRNA and protein levels of PD-L1 were detected to be elevated in both cell lines after FUT8 interference,and the mRNA levels of STAT1,JAK1,and IRF1 were further detected to be elevated(P<0.05).4.The mRNA expression levels of IL1b,TNF α,and CD86 were elevated in macrophages co-cultured with the two breast cancer cell lines after FUT8 interference,and the mRNA expression levels of ARG1,CD163,and CCL2 were decreased(P<0.05),and changes in the expression levels of IL10 were decreased(P<0.05)or not statistically different(P>0.05).CONCLUSION 1.After FUT8 interference with the Triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-468,the proliferation of both cell lines was inhibited and the cell cycle G0/G1 phase and S phase were shortened.2.FUT8 may promote the migration and invasive ability of both triple-negative breast cancer cell lines through epithelial mesenchymal transition.3.FUT8 interference increased PD-L1 mRNA and protein levels.4.Two triple-negative breast cancer cell lines after FUT8 interference slowed the progression of tumor-associated macrophages toward a tumor-promoting phenotype.