| Objective:To investigate the role of guanylate binding protein 2(GBP2)in immune infiltration and immune escape in clear cell renal cell carcinoma(ccRCC)and its molecular mechanism.Method:GEO,TIMER and GEPIA2 online tools were used to examin the differential expression of GBP2 in tumors.Survival analysis was performed using the online tool Kaplan-Meier plotter.UALCAN website was used to validate the clinicopathological features of GBP2.LinkedOmics and Metascape tools were used to analyze GBP2 co-expression-related genes and their GO and KEGG pathways.GeneMANIA database was used to analyze the relationship between GBP2 and its interacting genes.TIMER2.0 database was used to analyze the characteristics of immune infiltration of GBP2 in clear cell renal cell carcinoma(ccRCC)and its correlation with molecular markers of immune cells.The expression of GBP2 and PD-L1 and their correlation were analyzed using western blotting in the collected ccRCC samples and their paired adjacent tissues.Western Blotting was used to determine the expression of GBP2 in renal cancer cell lines.Focusing on the in vitro association between GBP2 and programmed death ligand 1(PD-L1),GBP2 was knocked down and overexpressed in Caki-1 and 786-O cells.RT-qPCR and Western blotting were used to explore the expression of PD-L1,signal transducer and transcription factor 1(STAT1)and its phosphorylated proteins.Secondly,we investigated the mechanism by which GBP2 overexpression regulated PD-L1 expression on the STAT1 pathway by exogenous use of STAT1 inhibitor and endogenous siRNA to knock down STAT1 gene,and co-immunoprecipitation was used to explore the interaction between GBP2 and STAT1.Result:The expression of GBP2 in ccRCC tissues was higher than that in adjacent tissues(p<0.05),and the overall survival rate of patients with high expression level was lower(p=0.0014).In addition,the expression level of GBP2 was significantly different in tissues of different stages and tumor grades.GO analysis showed that GBP2 and its related genes were mainly involved in the regulation of T cell activation and adaptive immune response.KEGG pathway analysis showed that GBP2 and its related genes were mainly enriched in natural killer cell-mediated cytotoxicity and T cell receptor signaling pathways.The protein interaction network results showed that GBP2 had a strong co-expression relationship with GBP1,STAT1,STAT2,IRF1 and other proteins.TIMER database showed that the expression level of GBP2 was positively correlated with different immune cells(B cells:cor=0.515,P=1.69*10-32;CD8+T cells:cor=0.596,P=1.69*10-43;CD4+T cells:cor=0.257,P=2.13*10-8;Macrophages:cor=0.305,P=4.33*10-11;Neutrophils:cor=0.516,P=1.50*10-32;Dendritic cells:cor=0.638,P=2.42*10-53).Meanwhile,in ccRCC,we found a strong positive correlation between GBP2 and gene markers related to CD8+T cells,T cells(general)and T cell exhaustion.GBP2 is overexpressed in renal cell carcinoma cell lines.RT-qPCR and western blot results showed that knockdown or overexpression of GBP2 reduced or up-regulated the expression of PD-L1,respectively.In addition,we found that GBP2 expression activated the STAT1 pathway.After STAT1 activity was inhibited by STAT1 inhibitor Fludarabine,the expression of PD-L1 was decreased,and the same results were obtained by knocking down STAT1 gene with siRNA,indicating that STAT1 is involved in the regulation of PD-L1 expression by GBP2.Co-immunoprecipitation showed that GBP2 could interact with STAT1.Conclusion:This study demonstrates that guanylate binding protein 2(GBP2)is up-regulated in clear cell renal cell carcinoma,and its high expression indicates poor prognosis.At the same time,the expression level of GBP2 is positively correlated with different immune cells,which is involved in the immune infiltration of renal clear cell carcinoma.In vitro culture,GBP2 promotes tumor cell immune escape by regulating PD-L1 expression through the STAT1 pathway,revealing that GBP2 may be a potential biomarker and immunotherapy target for ccRCC. |
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