Upregulation of ARNTL2 is associated with poor survival and immune infiltration in clear cell renal cell carcinoma

Abstract

Background: Aryl hydrocarbon receptor nuclear translocator like 2 (ARNTL2) is a member of the PAS superfamily. Previous studies explored the carcinogenic roles of transcription factor ARNTL2 in human malignancies. However, its roles in ccRCC have not been elucidated. This study sought to explore the roles of ARNTL2 in ccRCC and determine its correlations with tumor immunity. Methods: The expression of ARNTL2 was analyzed using the GEO, TCGA and GTEx database, and verified in ccRCC tissue samples and cell lines by qRT-PCR and western blot analysis. Kaplan–Meier survival curve analysis, Cox regression analysis (including univariate and multivariate analysis) was utilized to evaluate the prognostic values of ARNTL2. Potential biological mechanisms of ARNTL2 were explored using GSEA method. Colony formation and wound healing assays were conducted to explore the oncogenic role of ARNTL2 in ccRCC. ssGSEA and xCell algorithm were used to explore the correlation between ARNTL2 expression and tumor immune microenvironment (TIME). Results: ARNTL2 was significantly upregulated in ccRCC tissues and cell lines compared to normal kidney tissues and cell line. Enhanced expression of ARNTL2 was strongly linked to advanced clinical stage and unfavorable overall survival in ccRCC. ARNTL2 was determined as an independent prognostic marker through cox regression analysis. A prognostic nomogram was constructed to predict 1-, 3- and 5-year overall survival of ccRCC patients by integrating ARNTL2 expression with other clinicopathologic variables. GSEA analysis showed that focal adhesion, T cell receptor, cell cycle, and JAK-STAT signaling pathway were significantly enriched in high ARNTL2 samples. Silencing of ARNTL2 suppressed the colony formation ability and wound healing efficacy of ccRCC cell lines. xCell analysis showed that high expression level of ARNTL2 exhibited an immune infiltration status similar to CD8 + inflamed ccRCC subtype, which was characterized by high infiltration level of CD8 + T cell and high expression level of the immune escape biomarkers such as PD-L1, PD-L2, PD1 and CTLA4. Conclusion: ARNTL2 is an independent adverse predictor of ccRCC patient survival. High expression level of ARNTL2 is associated with immune infiltration, and may be a novel therapeutic target in ccRCC.