目的·探索RBX1（ring-box protein 1）在葡萄膜黑色素瘤（uveal melanoma，UVM）肿瘤细胞中对免疫相关基因的调控作用。方法·通过检索癌症基因组图谱（The Cancer Genome Atlas，TCGA）数据库分析RBX1在肿瘤中的表达水平以及与临床分期、生存预后的相关性。使用靶向RBX1的小干扰RNA（small interfering RNA，siRNA）分别在UVM细胞系92.1、OMM2.3和MEL290中瞬时敲低RBX1，并对瞬时敲低RBX1的92.1细胞进行转录组测序，分析siRBX1转染细胞与对照细胞的差异表达基因，并采用基因集富集分析（gene set enrichment analysis，GSEA）对差异基因进行分析，探究RBX1与肿瘤免疫相关基因的关系。在分析结果的基础上，通过实时荧光定量PCR（qPCR）分别检测瞬时敲低RBX1的92.1、OMM2.3和MEL290细胞系中信号转导与转录激活因子1（signal transducer and activator of transcription 1，STAT1）及其下游的CXC趋化因子配体9（C-X-C motif chemokine ligand 9，CXCL9）和CXCL10的mRNA表达水平，并通过Western blotting检测92.1细胞中STAT1及p-STAT1蛋白表达水平。在瞬时敲低RBX1的细胞系OMM2.3和MEL290中，分别加入5 nmol/L或10 nmol/L的STAT1抑制剂fludarabine，处理48 h后通过qPCR检测CXCL9和CXCL10的mRNA表达水平。结果·TCGA数据库分析表明：与正常组织相比，RBX1在多种肿瘤中高表达，在肾上腺皮质癌和UVM中显著高表达；且这2种肿瘤分期晚的患者，RBX1表达水平更高，同时RBX1表达水平高的患者的总生存期更短（均P<0.05）。对瞬时敲低RBX1的92.1细胞和对照细胞进行转录组测序，获得差异基因，并且GSEA结果显示，RBX1参与调控肿瘤免疫相关通路。热图分析显示RBX1敲低后STAT1表达水平上升。在92.1、OMM2.3和MEL290细胞系中，qPCR结果显示RBX1敲低后STAT1、CXCL9和CXCL10的mRNA表达水平上升，Western blotting结果显示在92.1细胞系中敲低RBX1后STAT1及p-STAT1表达水平上升。加入STAT1抑制剂后，OMM2.3和MEL290细胞系中的CXCL9和CXCL10 mRNA上调表达被抑制。结论·RBX1在UVM细胞中可能通过STAT1调控CXCL9和CXCL10的表达，参与肿瘤免疫调控。

Objective ·To investigate the role of RBX1 (ring-box protein 1) in the regulation of immune-related genes in uveal melanoma (UVM) tumor cells. Methods ·The Cancer Genome Atlas (TCGA) was searched to analyze the expression levels of RBX1 in tumors and the correlation with clinical stages and survival prognosis. RBX1 was transiently knocked down in the UVM cell lines, i.e., 92.1, OMM2.3 and MEL290 by using small interfering RNAs (siRNAs) targeting RBX1. RNA sequencing was performed on the 92.1 cells with transient knockdown of RBX1, and the differentially expressed genes between the siRBX1-transfected cells and control cells were analyzed by gene set enrichment analysis (GSEA) to investigate the relationship between RBX1 and tumor immune-related genes. Based on the results of the analysis, signal transducer and activator of transcription 1 (STAT1) and its downstream CXC chemokine ligand 9 (CXCL9) and CXCL10 mRNA expression levels were detected by real-time fluorescence quantitative PCR (qPCR) in 92.1, OMM2.3 and MEL290 cells with transient knockdown of RBX1, respectively. The protein expression levels of STAT1 and p-STAT1 in 92.1 cells were detected by Western blotting. The cell lines OMM2.3 and MEL290, in which RBX1 was transiently knocked down, were treated with 5 nmol/L or 10 nmol/L STAT1 inhibitor fludarabine for 48 h, and the mRNA expression levels of CXCL9 and CXCL10 were detected by qPCR. Results ·TCGA database analysis showed that RBX1 was highly expressed in a variety of tumors compared to the normal tissues, particularly in adrenocortical carcinoma and UVM. In addition, the patients with late stage of these two kinds of tumors had higher expression level of RBX1, and the patients with higher expression level of RBX1 had shorter overall survival time (P<0.05). RNA sequencing of 92.1 cells with transiently knocked down RBX1 and control cells revealed differential genes, and GSEA showed that RBX1 was involved in the regulation of tumor immune-related pathways. Heat map analysis showed an increase in STAT1 expression after RBX1 knockdown. In the 92.1, OMM2.3 and MEL290 cell lines, qPCR showed increases in STAT1, CXCL9 and CXCL10 mRNA expression after transient knockdown of RBX1, and Western blotting showed that STAT1 and p-STAT1 expression increased after knockdown of RBX1 in 92.1 cell lines. The increases of CXCL9 and CXCL10 mRNA in OMM2.3 and MEL290 cell lines were suppressed by STAT1 inhibitors. Conclusion ·RBX1 may regulate CXCL9 and CXCL10 expression via STAT1 in UVM cells and is involved in tumor immune regulation.