Toll-Like Receptors Serve as Biomarkers for Early Diagnosis and Prognosis Assessment of Kidney Renal Clear Cell Carcinoma by Influencing the Immune Microenvironment: Comprehensive Bioinformatics Analysis Combined With Experimental Validation

Abstract

Background: Toll-like receptors (TLRs) are important initiators of innate and acquired immune responses. However, its role in kidney renal clear cell carcinoma (KIRC) remains unclear.Methods: TLRs and their relationships with KIRC were studied in detail by ONCOMINE, UALCAN, GEPIA, cBioPortal, GeneMANIA, FunRich, LinkedOmics, TIMER and TRRUST. Moreover, we used clinical samples to verify the expressions of TLR3 and TLR4 in early stage of KIRC by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), flow cytometry (FC) and immunohistochemistry (IHC).Results: The expression levels of TLRs in KIRC were generally different compared with adjacent normal tissues. Moreover, the expressions of TLR3 and TLR4 elevated significantly in the early stage of KIRC. Overexpressions of TLR1, TLR3, TLR4 and TLR8 in KIRC patients were associated with longer overall survival (OS), while inhibition of TLR9 expression was related to longer OS. Additionally, overexpressions of TLR1, TLR3 and TLR4 in KIRC patients were associated with longer disease free survival (DFS). There were general genetic alterations and obvious co-expression correlation of TLRs in KIRC. The PPI network between TLRs was rather complex, and the key gene connecting the TLRs interaction was MYD88. The GO analysis and KEGG pathway analysis indicated that TLRs were closely related to adaptive immunity, innate immunity and other immune-related processes. RELA, NFKB1, IRF8, IRF3 and HIF1A were key transcription factors regulating the expressions of TLRs. What’s more, the expression levels of all TLRs in KIRC were positively correlated with the infiltration levels of dendritic cells, macrophages, neutrophils, B cells, CD4+ T cells and CD8+ T cells. Finally, the results of RT-qPCR, FC and IHC confirmed that TLR3 and TLR4 were significantly elevated in the early stage of KIRC.Conclusion: The occurrence and development of KIRC are closely related to TLRs, and TLRs have the potential to be early diagnostic biomarkers of KIRC and biomarkers for judging the prognosis and immune status of KIRC. This study may provide new insights into the selection of KIRC immunotherapy targets.