The Function Of Melanoma Tumour Cell-intrinsic PD-L1 In Epithelial-mesenchymal Transition Induced By TGF-β1

Objective: The aim of this study was to investigate the relationship between tumour cell-intrinsic programmed death receptor ligand-1(PD-L1)and transforming growth factor-β1(TGF-β1)in melanoma cells and the role of endogenous PD-L1 in epithelial mesenchymal transformation induced by TGF-β1.Methods: In vitro,murine melanoma cell lines B16-F0 and B16-F10 were treated with TGF-β1 and TGF-β1 inhibitors,respectively.The expression of PD-L1 was detected by flow cytometry and real-time quantitative PCR.After the melanoma cells were treated with PD-L1 inhibitor,the concentration of TGF-β1 in the culture supernatant was detected by ELISA and the expression of TGF-β1 in the cells was detected by RT-qPCR.Melanoma cells were transfected with PD-L1-shRNA lentivirus to construct a stable cell line silencing PD-L1.The effect of PD-L1 gene silencing on the expression of EMT related markers such as E-Cadherin and Fibronectin-1 induced by TGF-β1 was detected by RT-qPCR,and the effect of PD-L1 gene silencing on cell proliferation after TGF-β1 treatment was detected by CCK-8assay.In the in vivo experiment,C57BL/6 mice were subcutaneously inoculated with normal melanoma cells or PD-L1 silencing melanoma cells.The tumor formation of the two groups was monitored regularly.After the tumor tissue was dissected,the quantitative tissue was made into tissue homogenate,and the supernatant was taken for ELISA test to detect the level of TGF-β1.The tumor tissue sections were made to detect the expression of E-Cadherin,Fibronectin-1,Ki-67,PD-L1 and α-SMA protein by immunohistochemical method.RNA was extracted from tumor tissue and the expression of E-Cadherin and Fibronectin-1 was detected by RT-qPCR.Results: 1.TGF-β1 can up-regulate the expression of PD-L1 in melanoma cells,on the contrary,blocking TGF-β1 can down-regulate the expression of PD-L1 in melanoma cells.2.Blocking PD-L1 could inhibit the secretion and expression of TGF-β1 in melanoma cells.3.After PD-L1-silenced melanoma cells were stimulated by TGF-β1,the expression of epithelial-related marker E-Cadherin was increased,the expression of stromal related marker Fibronectin-1 was decreased,and the ability of cell proliferation was also decreased.4.In vivo experiment,the results showed that the tumor volume of mice inoculated with silenced PD-L1 melanoma cells decreased.The concentration of TGF-β1 in silenced PD-L1 tissue homogenate decreased.The expressions of E-Cadherin and Fibronectin-1 in silenced PD-L1 tumor tissues were increased and decreased,respectively.The expression of Ki-67 decreased in silenced PD-L1 tumor tissues.There was a co-localized expression relationship between PD-L1 and α-SMA in tumor tissues.RT-qPCR results showed that the expression of E-Cadherin increased and the expression of Fibronectin-1 decreased.Conclusion: There is a mutual regulation between TGF-β1 and tumour cell-intrinsic PD-L1 in melanoma cells,that is,TGF-β1 can induce the expression of PD-L1 and PD-L1 can induce the secretion and expression of TGF-β1.PD-L1 also participates in the regulation of epithelial mesenchymal transformation and cell proliferation induced by TGF-β1,which promotes the development of tumor.The co-localized expression of PD-L1 and α-SMA suggested that TGF-β1 might originate from tumor-associated fibroblasts.