Wang Song, Ma Xueyou, Ying Yufan, Sun Jiazhu, Yang Zitong, Li Jiangfeng, Jin Ke, Wang Xiao, Xie Bo, Zheng Xiangyi, Liu Ben, Xie Liping
Department of Urology, School of Medicine, The First Affiliated Hospital, Zhejiang University, 79 Qingchun Road, Hangzhou, 310003, Zhejiang, China.
Cancer Cell Int. 2021 Jul 3;21(1):341. doi: 10.1186/s12935-021-02046-z.
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.
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).
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.
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.
芳烃受体核转运蛋白样2（ARNTL2）是PAS超家族的成员。先前的研究探讨了转录因子ARNTL2在人类恶性肿瘤中的致癌作用。然而，其在ccRCC中的作用尚未阐明。本研究旨在探讨ARNTL2在ccRCC中的作用，并确定其与肿瘤免疫的相关性。
利用GEO、TCGA和GTEx数据库分析ARNTL2的表达，并通过qRT-PCR和蛋白质印迹分析在ccRCC组织样本和细胞系中进行验证。采用Kaplan-Meier生存曲线分析、Cox回归分析（包括单变量和多变量分析）来评估ARNTL2的预后价值。使用GSEA方法探索ARNTL2的潜在生物学机制。进行集落形成和伤口愈合试验以探讨ARNTL2在ccRCC中的致癌作用。使用单样本基因集富集分析（ssGSEA）和xCell算法来探索ARNTL2表达与肿瘤免疫微环境（TIME）之间的相关性。
与正常肾组织和细胞系相比，ARNTL2在ccRCC组织和细胞系中显著上调。ARNTL2表达增强与ccRCC的晚期临床分期和不良总生存期密切相关。通过Cox回归分析确定ARNTL2为独立的预后标志物。构建了一个预后列线图，通过将ARNTL2表达与其他临床病理变量相结合来预测ccRCC患者1年、3年和5年的总生存期。GSEA分析表明，在高ARNTL2样本中，粘着斑、T细胞受体、细胞周期和JAK-STAT信号通路显著富集。沉默ARNTL2可抑制ccRCC细胞系的集落形成能力和伤口愈合效果。xCell分析表明，ARNTL2的高表达水平表现出与CD8+炎症性ccRCC亚型相似的免疫浸润状态，其特征是CD8+T细胞浸润水平高，以及免疫逃逸生物标志物如PD-L1、PD-L2、PD1和CTLA4的高表达水平。
ARNTL2是ccRCC患者生存的独立不良预测指标。ARNTL2的高表达水平与免疫浸润相关，可能是ccRCC的一个新的治疗靶点。
