Functional Mechanism Of APLNR In IFN-γ-mediated PD-L1 Expression

Purpose:Our previous study found that APLNR inhibited EMT in nasopharyngeal carcinoma through the Akt/mTOR signaling pathway,but it is unclear whether APLNR has other downstream molecules and their corresponding functions.It has been reported that IFN-γ can mediate the expression of PD-L1 in tumor cells,and this paper mainly wants to explore whether APLNR is involved in IFN-γ mediates the expression of PD-L1 and the molecular functional mechanisms of regulation.Methods:Human nasopharyngeal carcinoma cell lines(5-8F,HNE2)with overexpression and interference of APLNR were established.The effects of APLNR on proliferation and apoptosis of nasopharyngeal carcinoma cells were investigated by plate cloning,flow cytometry and Hoechst staining.WB confirmed the mechanism of APLNR regulating nasopharyngeal carcinoma cell apoptosis.In nasopharyngeal carcinoma cells,the expression of p-JAK1,PD-L1,p-STAT1 stimulated by IFN-γ was detected.Then Upadacitinib(JAK1 inhibitor)and Fludarabine(STAT1 inhibitor)were added to detect PD-L1 protein changes in nasopharyngeal carcinoma cells.WB was used to detect the expression levels of p-JAK1,p-STAT1 and PD-L1 after overexpression and interference of APLNR.The interaction and co-location of APLNR and JAK1 were detected by immunoprecipitation and immunofluorescence assay.Finally,nasopharyngeal carcinoma cells overexpressing APLNR were co-cultured with human peripheral blood lymphocytes.Results:Plate colony experiments,flow cytometry,and Hoechst staining showed that APLNR could significantly inhibit nasopharyngeal carcinoma cell survival,increase apoptotic rate and apoptotic body formation.WB results showed that Cleaved Caspase-3 expression was increased or decreased by overexpression or interference of APLNR,respectively,and IFN-γ significantly up-regulated the expression of PD-L1,p-JAK1,and p-STAT1 in nasopharyngeal carcinoma cells.JAK1 and STAT1 inhibitors can reverse this effect.WB results showed that the expression of PD-L1,p-JAK1 and p-STAT1 decreased after overexpression of APLNR,while the expression of p-JAK1,p-STAT1 and PD-L1 increased after interference of APLNR.Immunoprecipitation and cell immunofluorescence experiments demonstrated that APLNR could directly interact with JAK1.Co-culture experiments of nasopharyngeal carcinoma cells and lymphocytes with overexpression of APLNR showed that APLNR could enhance the ability of lymphocytes to inhibit tumor cell proliferation.Conclusions:1、APLNR inhibited proliferation and promoted apoptosis of nasopharyngeal carcinoma cells;2、APLNR inhibited IFN-γup-regulated PD-L1 expression by inactivating JAK1/STAT1 signaling pathway through its interaction with JAK1.