Role And Mechanism Of Inflammation Regulated PD-L1 In The Tumorgenesis And Progression Of Glioblastoma

Objective: Glioblastoma(GBM)is the most malignant primary glioma.Conventional treatment methods include surgery,radiotherapy and chemotherapy have limited curative effect on the tumor.Patients have high mortality and poor prognosis.With the deepening of molecular biology research,molecular targeted therapy has opened a new era of tumor therapy.Programmed death ligand 1(PD-L1),as a member of B7 family,has been proved to play an important role in plays an important role in tumor immune escape process.Studies have found that PD-L1 is specifically expressed in GBM tissues and cells,but there are few studies on inflammation regulating PD-L1 in GBM.Therefore,We investigated GBM cell lines and primary tumor cells of patients.We analyzed the effect of IFN-γ as an inflammatory mediator on PD-L1 expression in GBM cells.We explored the specific mechanism of IFN-γ regulating PD-L1 expression and the role of PD-L1 in the tumorgenesis and progression of GBM.The aim was to provide experimental basis for GBM therapy strategy targeting PD-L1.Methods:(1)Western blot and flow cytometry were used to detect the expression of PD-L1 in GBM cell lines and primary tumor cells obtained from GBM patients.(2)After IFN-γ(50ng/mL)was used to stimulate GBM cells,the expression of PD-L1 was detected by quantitative real-time polymerase chain reaction(qRT-PCR),flow cytometry and Western blot.(3)Short hairpin RNA(shRNA)was used to knock down the expression of PD-L1 in GBM cells.(4)Cell counting kit-8(CCK-8)experiment and Carboxyfloresin Succinimidyl Aminotester(CFSE)labeling method were used to detect the proliferation ability of GBM cells.(5)The migration ability of GBM cells was tested by scratch healing experiment.(6)Western blot was used to detect the expression of related protein molecules involved in the proliferation,invasion and migration of GBM cells.(7)Western blot was used to detect the expression of signal transduction pathway related molecular proteins involved in IFN-γ regulated PD-L1.(8)SPSS 22.0 and Graphpad prism 8.0 software were used to analyze the experimental data.All data were verified by three repeated experiments.Student’s t-test was used to compare two groups.p<0.05 was considered statistically significant.Results:(1)PD-L1 was expressed in GBM cell lines and primary tumor cells.(2)After stimulated with IFN-γ,the expression of PD-L1 increased at mRNA,surface protein and total protein.(3)IFN-γ upregulated the expression of PD-L1 in GBM cells by activating ERK signaling pathway.(4)shRNA was used to knock down the expression of PD-L1 in GBM cells.(5)After PD-L1 was knocked down,the proliferation and migration of GBM cells were inhibited.After stimulated with IFN-γ,the proliferation and migration of GBM cells remained the same.(6)After PD-L1 was knocked down,the expression of CDK4,CDK6,MMP2 and Vimentin in GBM cells was inhibited.After stimulated with IFN-γ,the expression of CDK4,CDK6,MMP2 and Vimentin in GBM cells were inhibited.Conclusions:(1)IFN-γ upregulated the expression of PD-L1 in GBM cells by activating ERK signaling pathway.(2)PD-L1 promoted the proliferation and migration of GBM cells via regulating the expression of CDK4,CDK6,MMP2 and Vimentin.(3)IFN-γ had no effect on the proliferation and migration of GBM cells.