Study On PD-L1 Core Fucosylation Associated With Rg3 In Triple-Negative Breast Cancer

Objective: PD-L1 expressed by tumor cells can bind with PD-1 expressed by tumor infiltrating lymphocytes,which promotes tumor escape from the killing of lymphocytes and immune escape,resulting in tumorigenesis and progression.However,the role of PDL1 glycosylation level in breast cancer is still unclear,especially in the core fucosylation level.Studies have shown that PD-L1 has α1,6-fucosylation,that is,core fucosylation.Fucosyltransferase 8(FUT8)is the key enzyme responsible for catalyzing the formation of core fucosylation,and its expression level can affect the level of protein glycosylation.At present,the exploration of core fucosylation level of PD-L1 in breast cancer cells is not clear.This study in order to explore the relationship between PD-L1 and FUT8;to determine the effect and mechanism of core fucosylation of PD-L1 on the proliferation,migration and immune escape function of triple negative breast cancer cell line MDAMB-231;Meanwhile,Ginsenoside Rg3 was used as an auxiliary treatment in our study,so as to provide theoretical basis and new strategies for the treatment of breast cancer.and to explore whether Ginsenoside Rg3 can affect the level of PD-L1 glycosylation,so as to assist in the treatment of breast cancer.Methods: Western blot,immunofluorescence and real-time qPCR were used to detect the expression of PD-L1 and FUT8 in MCF-7,MDA-MB-231 and BT549 cells.The glycosylation level of PD-L1 was determined by LCA fluorescence staining,lectin blot and laser confocal microscopy.The proliferation of MDA-MB-231 cells was detected by colony forming assay.Scratch test was used to detect the migration ability of PD-L1 cells.The cytotoxic effect of cytotoxic T cells on PD-L1 core fucosylation reduced cancer cells was detected by crystal violet staining and flow cytometry.The expressions of PI3Kp110β,p-Akt and Akt were detected by Western blot and immunofluorescence.LY290004,an inhibitor of PI3 K pathway,was used to detect the expression of PD-L1 to speculate the role of this pathway in regulating the expression of PD-L1.Results: After preliminary screening of PD-L1 and FUT8 in three cell lines,MDAMB-231,a triple negative breast cancer cell line with high expression of PD-L1 and FUT8,was selected as the model of follow-up experiment.We found that:1.In MDA-MB-231 cells,FUT8 siRNA transfection down regulated the expression of FUT8 at protein and m RNA levels.In the three breast cancer cells,MDA-MB-231 cell line had a high level of core fucosylation,and FUT8 siRNA reduced the overall level of protein core fucosylation,and further confirmed that it could affect the level of core fucosylation on PD-L1.Furthermore,we found that the level of PD-L1 protein was inhibited after FUT8 gene knockdown.Lectin imprinting showed that there was core fucose at the same molecular weight as PD-L1.Immunofluorescence results showed that fut8 gene knockdown affected the expression of PD-L1 protein and reduced core fucose.2.The results of functional experiments showed that FUT8 knockdown reduced the core fucosylation of PD-L1,and the proliferation and migration ability of MDA-MB-231 cells were decreased compared with the control group,but the effect was not as obvious as that of direct knockdown of PD-L1.At the same time,T cell cytotoxicity test and apoptosis results showed that reducing the core fucosylation of PD-L1 can enhance the toxicity of T cells to cancer cells,which can well inhibit the immune escape of tumor cells.3.Rg3 can inhibit the proliferation of cancer cells and promote apoptosis.The combination of Rg3 and FUT8 siRNA can further reduce the proliferation and migration ability of cancer cells,which is more significant than knockdown of PD-L1 alone.At the same time,the killing effect of cytotoxic T cells on MDA-MB-231 was further enhanced.Rg3 treatment or FUT8 siRNA inhibited the activation of PI3K/Akt signaling pathway,while FUT8 siRNA transfection combined with Rg3 further reduced the protein expression levels of PI3Kp110β,p-Akt and PD-L1.4.After LY290004 inhibited PI3 K pathway,the expression level of PD-L1 protein decreased.These results indicate that FUT8 and Rg3 regulate the expression of PD-L1 through PI3K/Akt signaling pathway,thereby affecting the proliferation,migration and sensitivity of cancer cells to T cells.Conclusion: In conclusion,our findings suggest that FUT8 can regulate the core fucosylation level of PD-L1 protein in MDA-MB-231 triple negative breast cancer cells.Down regulating the core fucosylation of PD-L1 protein can inhibit the proliferation and migration of breast cancer cells,and increase the sensitivity of cancer cells to T cells.Rg3 and si-FUT8 transfection can effectively inhibit the proliferation and migration of MDAMB-231 cells and restore the immune function of T cells by inhibiting PI3K/Akt pathway and down regulating the expression of PD-L1.