Identification And Validation Of Immune-related Biomarkers In The Tumor Microenvironment Of Clear Cell Renal Cell Carcinoma

Object:Clear cell renal cell carcinoma(cc RCC)is a common and highly malignant kidney tumor.Unfortunately,due to a lack of early warning signs,nearly 40%of patients were in advanced stages of disease at the time of diagnosis.What’s worse,advanced patients are insensitive to radiotherapy and chemotherapy,which results in a 5-year survival rate of less than 10%.According to recent studies,the tumor microenvironment is an important factor affecting the efficacy of immunotherapy,and the tumor microenvironment of cc RCC was highly infiltrated by immune cells,suggesting that the immune process may play an important role in the occurrence and development of cc RCC.However,the specific mechanism is not clear and lacks corresponding biomarkers.Thus it is necessary to explore biomarkers associated with the tumor immune microenvironment to provide a theoretical basis for the treatment of cc RCC.Methods:(1)We downloaded cc RCC RNA expression data from the Gene Expression Omnibus,which contains 144 samples.Differentially expressed genes were identified between tumor tissues and adjacent noncancerous tissues.The differentially expressed genes were analyzed using weighted correlation network analysis(WGCNA)and the deconvolution algorithm that quantifies the cellular composition of immune cells(CIBERSORT).Then,we filtered out a hub module that has the highest correlation with tumor immune infiltrating cells.Finally,hub genes were identified according to the hub module and protein-protein interaction network.(2)We downloaded cc RCC RNA expression data from the TCGA database.Differential expression at the transcriptome level of hub genes was validated by GEPIA and Oncomine databases.Also,the differential expression at the protein level was analyzed by the HPA database.Furthermore,the TIMER and TISIBI database were applied to validate the correlation between hub genes and tumor-infiltrating immune cells in the tumor surrounding environment.Finally,the Kaplan Meier plotter database was applied to filter out the hub genes with prognostic value.(3)We used 769-P,a human renal cell adenocarcinoma cell line,as an in vitro study.Cell migration ability was explored by cell scratch assay,while the cell invasion ability was explored by Transwell cell invasion assay.Thereby validating the basic biological functions of prognostic biomarkers in cc RCC.Results:(1)WGCNA analysis identified the yellow module as a hub module correlating with CD8~+T cells.Gene in the yellow module mainly functioned in signaling pathways such as PD-1/PD-L1 and adaptive immune response.(2)Initial screening resulted in 13 hub genes that were highly correlated with CD8~+T cells,including PRF1,CD27,CXCL10,CD3E,GBP1,CCL5,GBP5,GZMA,CD2,GBP2,IRF1,CXCL9,and CXCR6.(3)Except for IRF1,all 12 genes were differentially expressed and statistically significant between the tumor and control groups(P<0.05).Also,12 genes were highly correlated with tumor immune infiltrating cells.What’s more,high expression of CCL5,CD27,GBP2,GBP5 and GZMA genes was found to be negatively associated with the prognosis of patients with cc RCC through survival analysis(HR>1,P<0.05).(4)The results of cell scratch showed that the percentage of wound healing in the GBP2 overexpression group was significantly higher than that in the control group(P<0.05).Meanwhile,the results of cell invasion showed that the number of cell invasions in the GBP2 overexpression group was significantly higher than that in the control group(P<0.05).Conclusions:PRF1,CD27,CXCL10,CD3E,GBP1,CCL5,GBP5,GZMA,CD2,GBP2,CXCL9,CXCR6 may be immune-related biomarkers.Among them,CCL5,CD27,GBP2,GBP5,GZMA were immune-related prognosis biomarkers.Cellular assays initially validated that GBP2 overexpression promotes cc RCC migratory and invasive capacity,suggesting that GBP2 may be a potential biomarker and therapeutic target for cc RCC associated with CD8~+T cell immune infiltration.