RBX1通过STAT1调控葡萄膜黑色素瘤免疫相关基因

doi:10.3969/j.issn.1674-8115.2023.06.007

上海交通大学学报（医学版） ›› 2023, Vol. 43 ›› Issue (6): 709-717.doi: 10.3969/j.issn.1674-8115.2023.06.007

RBX1通过STAT1调控葡萄膜黑色素瘤免疫相关基因

周晓雯(), 李倩, 张哲, 沈键锋(), 范先群()

上海交通大学医学院附属第九人民医院眼科，上海市眼眶病眼肿瘤重点实验室，上海 200011

收稿日期:2023-03-16 接受日期:2023-05-15 出版日期:2023-06-28 发布日期:2023-06-28
通讯作者: 沈键锋,范先群 E-mail:hiumanchow@163.com;jfshen@shsmu.edu.cn;fanxianqun@126.com
作者简介:周晓雯（1997—），女，硕士生；电子信箱：hiumanchow@163.com。
基金资助:
国家自然科学基金(81972667);上海市重点实验室资助项目(20DZ2270800)

RBX1 regulates uveal melanoma immune-related genes via STAT1

ZHOU Xiaowen(), LI Qian, ZHANG Zhe, SHEN Jianfeng(), FAN Xianqun()

Department of Ophthalmology, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Orbital Diseases and Ocular Oncology, Shanghai 200011, China

Received:2023-03-16 Accepted:2023-05-15 Online:2023-06-28 Published:2023-06-28
Contact: SHEN Jianfeng,FAN Xianqun E-mail:hiumanchow@163.com;jfshen@shsmu.edu.cn;fanxianqun@126.com
Supported by:
National Natural Science Foundation of China(81972667);Program of Shanghai Key Laboratory Funding(20DZ2270800)

摘要/Abstract

摘要：

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。

关键词: 葡萄膜黑色素瘤, RBX1, 肿瘤免疫, 信号转导与转录激活因子1

Abstract:

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.

Key words: uveal melanoma (UVM), ring-box protein 1 (RBX1), tumor immunity, signal transducer and activator of transcription 1 (STAT1)

中图分类号:

R739.7

周晓雯, 李倩, 张哲, 沈键锋, 范先群. RBX1通过STAT1调控葡萄膜黑色素瘤免疫相关基因[J]. 上海交通大学学报（医学版）, 2023, 43(6): 709-717.

ZHOU Xiaowen, LI Qian, ZHANG Zhe, SHEN Jianfeng, FAN Xianqun. RBX1 regulates uveal melanoma immune-related genes via STAT1[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(6): 709-717.

图/表 8

表1 siRNA序列

Tab 1 siRNA sequences

Primer name	Sequence (5'→3')
siRBX1-1	GGGAUAUUGUGGUUGAUAAtt
siRBX1-2	CAGACCGUGUGUUUCCAAAtt

表2 qPCR检验的引物序列

Tab 2 Primer sequences for qPCR analysis

Gene	Forward (5'→3')	Reverse (5'→3')
β-actin	CATGTACGTTGCTATCCAGGC	CTCCTTAATGTCACGCACGAT
RBX1	TTGTGGTTGATAACTGTGCCAT	GACGCCTGGTTAGCTTGACAT
STAT1	CAGCTTGACTCAAAATTCCTGGA	TGAAGATTACGCTTGCTTTTCCT
CXCL9	TGAGAAAGGGTCGCTGTTCC	TCAAACTGCTTGGCTCACCA
CXCL10	GCTTCCAAGGATGGACCACA	GCAGGGTCAGAACATCCACT

图1 RBX1 在肿瘤中的表达水平及对患者生存的影响Note： A. Search for RBX1 expression in different tumors in GEPIA database. B. Overall survival heat map (above) and disease free survival heat map (below) of RBX1 in patients with various tumors. TPM—transcript per million; ACC—adrenocortical carcinoma; BLCA—bladder urothelial carcinoma; BRCA—breast invasive carcinoma; CESC—cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL—cholangiocarcinoma; COAD—colon adenocarcinoma; DLBC—lymphoid neoplasm diffuse large B-cell lymphoma; ESCA—esophageal carcinoma; GBM—glioblastoma multiforme; HNSC—head and neck squamous cell carcinoma; PAAD—pancreatic adenocarcinoma; PRAD—prostate adenocarcinoma; READ—rectum adenocarcinoma; SARC—sarcoma; SKCM—skin cutaneous melanoma; STAD—stomach adenocarcinoma; TGCT—testicular germ cell tumor; THYM—thymoma; UCEC—uterine corpus endometrial carcinoma; UCS—uterine carcinosarcoma; PCPG—pheochromocytoma and paraganglioma; KICH—kidney chromophobe; KIRC—kidney renal clear cell carcinoma; KIRP—kidney renal papillary cell carcinoma; LAML—acute myeloid leukemia; LGG—brain lower grade glioma; LIHC—liver hepatocellular carcinoma; LUAD—lung adenocarcinoma; LUSC—lung squamous cell carcinoma; MESO—mesothelioma; OV—ovarian serous cystadenocarcinoma; THCA—thyroid carcinoma; UVM—uveal melanoma.

Fig 1 Expression of RBX1 in tumors and its effect on patients' survival

图2 RBX1 表达水平与UVM和ACC患者分期及总生存期的关系Note： A. RBX1 expression levels in different stages of UVM patients (left) and overall survival curves of high expression and low expression patients (right). B. RBX1 expression levels in different stages of ACC patients (left) and overall survival curves of high expression and low expression patients (right).

Fig 2 Relationships of RBX1 expression level with stages and overall survivals of UVM and ACC patients

图3 转染siRNA后UVM细胞系中 RBX1 mRNA及蛋白的表达变化Note：A. Expression of RBX1 in 92.1 cell line. B. Expression of RBX1 in OMM2.3 cell line. C. Expression of RBX1 in Mel290 cell line. ①P=0.011, ②P=0.016, ③P=0.020.

Fig 3 Expression changes of RBX1 mRNA and protein in UVM cell lines transfected with siRNA

图4 92.1细胞系中敲低 RBX1 对免疫相关通路的影响Note： A. Differential gene enrichment analysis after siRBX1-1 (left) or siRBX1-2 (right) intervention compared with siNC. B. Differential genes enriched in the interferon-mediated signaling pathway after siRBX1-1 (left) or siRBX1-2 (right) intervention compared with siNC, and the associated differential genes heat map. NES—normalized enrichment score; STAT2—signal transducer and activator of transcription 2; IL10RB—interleukin 10 receptor subunit β; IFNAR1—interferon α and β receptor subunit 1; IRF9—interferon regulatory factor 9; JAK1—Janus kinase 1; IFNGR2—interferon γ receptor 2; TYK2—tyrosine kinase 2; IFNE—interferon ε; IFNK—interferon κ; IFNGR1—interferon γ receptor 1; PIK3R1—phosphoinositide-3-kinase regulatory subunit 1; IFNA1—interferon α1; IFNA10—interferon α10; IFNA13—interferon α13; IFNA14—interferon α14; IFNA16—interferon α16; IFNA17—interferon α17; IFNA2—interferon α2; IFNA21—interferon α21; IFNA4—interferon α4; IFNA5—interferon α5; IFNA6—interferon α6; IFNA7—interferon α7; IFNA8—interferon α8; IFNB1—interferon β1; IFNG—interferon γ; IFNL1—interferon λ1; IFNL2—interferon λ2; IFNL3—interferon λ3; IFNLR1—interferon λ receptor 1; PIK3CA—phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α; IFNW1—interferon ω1; JAK2—Janus kinase 2; IFNAR2—interferon α and β receptor subunit 2; PRKCA—protein kinase Cα.

Fig 4 Effect of RBX1 knockdown on immune-related pathways in 92.1 cell line

图5 RBX1 对UVM细胞中 STAT1 及相关基因的表达调控作用Note：A. mRNA expression of STAT1, CXCL9, and CXCL10 in 92.1 cell line after siRBX1 intervention detected by qPCR. B. mRNA expression of STAT1, CXCL9, and CXCL10 in OMM2.3 cell line after siRBX1 intervention detected by qPCR. C. mRNA expression of STAT1, CXCL9, and CXCL10 in MEL290 cell line after siRBX1 intervention detected by qPCR. D. STAT1 and p-STAT1 protein expression levels in 92.1 cell line after siRBX1 intervention detected by Western blotting. ①P=0.029, ②P=0.009, ③P=0.048, ④P=0.045, ⑤P=0.017, ⑥P=0.006, ⑦P=0.028, ⑧P=0.047, ⑨P=0.001, ⑩P=0.000, 11P=0.018, 12P=0.043, 13P=0.019, 14P=0.002, 15P=0.025, 16P=0.012, 17P=0.033.

Fig 5 Regulation of RBX1 on STAT1 and related genes expressions in UVM cell lines

图6 STAT1抑制剂对 RBX1 敲低后的UVM细胞中 CXCL9 和 CXCL10 mRNA表达的影响Note： The RBX1 knockdown cell lines of OMM2.3 (A) and MEL290 (B) were treated with 5 nmol/L and 10 nmol/L fludarabine. The samples were collected after 48 h of dosing and qPCR was performed. ①P=0.002, ②P=0.003, ③P=0.041, ④P=0.006, ⑤P=0.004, ⑥P=0.007, ⑦P=0.000, ⑧P=0.027, ⑨P=0.012, ⑩P=0.008, 11P=0.035.

Fig 6 Effect of STAT1 inhibitor on the mRNA expression of CXCL9 and CXCL10 in UVM cell lines after RBX1 knockdown

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RBX1通过STAT1调控葡萄膜黑色素瘤免疫相关基因

RBX1 regulates uveal melanoma immune-related genes via STAT1

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