APOBEC3B调控葡萄膜黑色素瘤复制应激的研究

doi:10.3969/j.issn.1674-8115.2022.08.008

上海交通大学学报（医学版） ›› 2022, Vol. 42 ›› Issue (8): 1034-1044.doi: 10.3969/j.issn.1674-8115.2022.08.008

• 论著 · 基础研究 • 上一篇

APOBEC3B调控葡萄膜黑色素瘤复制应激的研究

宗春燕(), 何杰, 张哲, 贾仁兵, 沈键锋()

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:2022-03-15 接受日期:2022-06-25 出版日期:2022-07-25 发布日期:2022-07-25
通讯作者: 沈键锋 E-mail:zongchunyan@sjtu.edu.cn;jfshen@shsmu.edu.cn
作者简介:宗春燕（1996—），女，硕士生；电子信箱：zongchunyan@sjtu.edu.cn。
基金资助:
国家自然科学基金面上项目(81972667);上海高校特聘教授（东方学者）项目(TP2018046);上海市教育委员会高峰高原学科建设计划(20191817)

Role of APOBEC3B in regulating replication stress of uveal melanoma

ZONG Chunyan(), HE Jie, ZHANG Zhe, JIA Renbing, SHEN Jianfeng()

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:2022-03-15 Accepted:2022-06-25 Online:2022-07-25 Published:2022-07-25
Contact: SHEN Jianfeng E-mail:zongchunyan@sjtu.edu.cn;jfshen@shsmu.edu.cn
Supported by:
General Program of National Natural Science Foundation of China(81972667);Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning(TP2018046);Shanghai Municipal Education Commission—Gaofeng Clinical Medicine Grant Support(20191817)

摘要/Abstract

摘要：

目的·研究载脂蛋白B mRNA编辑酶催化亚基3B（apolipoprotein B mRNA editing enzyme catalytic subunit 3B，APOBEC3B）在葡萄膜黑色素瘤（uveal melanoma，UM）中的生物学功能以及对药物治疗敏感性的影响。方法·应用免疫组织化学染色和Western blotting，分别检测眼部黑色素瘤［UM和结膜黑色素瘤（conjunctival melanoma，CM）］组织和细胞中APOBEC3B蛋白的表达水平。通过检索癌症基因组图谱（The Cancer Genome Atlas，TCGA）数据库，分析UM中APOBEC3B表达水平与预后的关联。使用CRISPR-Cas9质粒APOBEC3B-sgRNA敲除UM细胞系OMM2.3中APOBEC3B基因，构建APOBEC3B敲除的OMM2.3稳转细胞系sgAPOBEC3B以及对照细胞系Vector；并通过克隆形成实验，探索APOBEC3B敲除对OMM2.3细胞增殖的影响。同时，通过APOBEC3B-shRNA和空载质粒建立APOBEC3B敲低的OMM2.3细胞shAPOBEC3B和对照细胞shCtrl，对其进行转录组测序（RNA sequencing，RNA-seq），检测差异基因，并通过基因集富集分析（gene set enrichment analysis，GSEA）揭示APOBEC3B调控的下游通路。通过免疫荧光染色鉴定下游通路的关键蛋白。通过应用通路靶向药物处理，检测APOBEC3B对UM药物治疗敏感性的影响。结果·免疫组织化学染色结果表明眼部黑色素瘤（UM和CM）组织相较于良性色素痣组织APOBEC3B表达更高。Western blotting也表明包括OMM2.3细胞在内的大多数眼部黑色素瘤细胞相较于正常对照细胞（视网膜色素上皮细胞）APOBEC3B蛋白表达水平更高。并且TCGA数据库分析表明，APOBEC3B高表达的UM患者总生存期（P=0.032）和无病生存期（P=0.000）更短。然而APOBEC3B 敲除并不影响OMM2.3细胞克隆形成的能力，APOBEC3B敲低也没有造成细胞周期的显著改变。shAPOBEC3B和shCtrl细胞系的RNA-seq差异基因富集分析结果显示，APOBEC3B参与调控OMM2.3细胞的复制应激相关通路，并且热图分析也显示APOBEC3B敲低后DNA复制应激相关通路基因表达发生改变。免疫荧光染色结果显示，APOBEC3B敲低后复制应激通路关键靶标磷酸化的复制蛋白A 32 kDa亚基（replication protein A 32 kDa subunit，RPA32）水平降低。在对APOBEC3B敲低的OMM2.3细胞和对照细胞的药物敏感性实验中，发现相较于shAPOBEC3B细胞，shCtrl细胞对细胞周期检测点激酶1（cell cycle checkpoint kinase 1，CHK1）抑制剂的敏感性更高。结论·APOBEC3B参与调控OMM2.3细胞复制应激相关通路，且APOBEC3B表达的OMM2.3细胞对CHK1抑制剂的处理更为敏感。

关键词: 载脂蛋白B mRNA编辑酶催化亚基3B（APOBEC3B）, 葡萄膜黑色素瘤, 复制应激, 细胞周期检测点激酶1（CHK1）抑制剂

Abstract:

Objective ·To explore the biological function of apolipoprotein B mRNA editing enzyme catalytic subunit 3B (APOBEC3B) in uveal melanoma (UM) and its impact on drug sensitivity. Methods ·The expression of APOBEC3B in ocular melanoma [UM and conjunctival melanoma (CM)] tissues and cell lines were verified by immunohistochemical staining and Western blotting, respectively. The association between APOBEC3B expression and prognosis in UM was analyzed by searching The Cancer Genome Atlas (TCGA) database. In order to construct the OMM2.3-APOBEC3B knockout cell line sgAPOBEC3B and control cell line Vector, the CRISPR-Cas9 plasmid APOBEC3B-sgRNA targeting APOBEC3B gene was designed. Colony formation assay was used to examine the effect of APOBEC3B knockout on OMM2.3 cell proliferation. At the same time, plasmid APOBEC3B-shRNA targeting APOBEC3B gene was designed to construct the OMM2.3-APOBEC3B knockdown cell line shAPOBEC3B and control cell line shCtrl. Differential genes of shAPOBEC3B and shCtrl cell line were detected by RNA sequencing (RNA-seq), the result of which was analyzed by gene set enrichment analysis (GSEA) in order to find the downstream pathways APOBEC3B involved in. Key downstream protein of downstream pathway was identified by immunofluorescence staining. Then targeted drugs of this pathway were applied to examine the effect of APOBEC3B on drug sensitivity in UM treatment. Results ·Immunohistochemical staining results showed that ocular melanoma (UM and CM) tissues had higher APOBEC3B expression than benign pigmented nevus tissues. Western blotting also showed that most ocular melanoma cells, including OMM2.3 cells, expressed higher levels of APOBEC3B compared to normal control cells, retinal pigment epithelium cells. Also, TCGA database analysis showed that UM patients with higher APOBEC3B expression had shorter overall survival (P=0.032) and disease-free survival (P=0.000). However, APOBEC3B knockout did not affect the colony formation ability of OMM2.3 cells, nor did APOBEC3B knockdown cause significant changes in the cell cycle. Differential gene enrichment analysis on the RNA-seq results of shAPOBEC3B and shCtrl cells showed that APOBEC3B regulated replication stress-related pathways in OMM2.3 cells. The heat map also showed that the gene expression of DNA replication stress-related pathways altered after APOBEC3B knockdown. Immunofluorescence staining showed that the level of phosphorylated replication protein A 32 kDa subunit (RPA32), a key target of replication stress pathway, was decreased after APOBEC3B knockdown. In drug sensitivity experiments on shAPOBEC3B and shCtrl OMM2.3 cells, it was found that OMM2.3 cells with APOBEC3B expression were more sensitive to cell cycle checkpoint kinase 1 (CHK1) inhibitor. Conclusion ·APOBEC3B is involved in regulating the replication stress-related pathways of OMM2.3 cells, and APOBEC3B-expressing OMM2.3 cells are more sensitive to the treatment of CHK1 inhibitor.

Key words: apolipoprotein B mRNA editing enzyme catalytic subunit 3B (APOBEC3B), uveal melanoma (UM), replication stress, cell cycle checkpoint kinase 1 (CHK1) inhibitor

中图分类号:

R773.4

宗春燕, 何杰, 张哲, 贾仁兵, 沈键锋. APOBEC3B调控葡萄膜黑色素瘤复制应激的研究[J]. 上海交通大学学报（医学版）, 2022, 42(8): 1034-1044.

ZONG Chunyan, HE Jie, ZHANG Zhe, JIA Renbing, SHEN Jianfeng. Role of APOBEC3B in regulating replication stress of uveal melanoma[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(8): 1034-1044.

图/表 5

表1 qRT-PCR 检测相关基因的引物序列

Tab 1 Genes and primer sequences used for qRT-PCR analysis

Primer name	Forward （5'→3'）	Reverse （5'→3'）
APOBEC3B	CGCCAGACCTACTTGTGCTAT	CATTTGCAGCGCCTCCTTAT
GAPDH	AGGTCGGTGTGAACGGATTTG	TGTAGACCATGTAGTTGAGGTCA
CCNH	TGTTCGGTGTTTAAGCCAGCA	TCCTGGGGTGATATTCCATTACT
HEXB	CTCGCCCCGGAGAACTTCTA	GAAAGCATCACACTCTGACTGA
IGFBP3	AGAGCACAGATACCCAGAACT	GGTGATTCAGTGTGTCTTCCATT
DCT	CTTGGGCTGCAAAATCCTGC	CAGCACTCCTTGTTCACTAGG
CDKN1C	GCGGCGATCAAGAAGCTGT	GCTTGGCGAAGAAATCGGAGA
EIF3C	ATGTCGCGGTTTTTCACCAC	GCGGACAACTCTCTTGGTATCT

图1 APOBEC3B在肿瘤中的表达情况以及对UM患者生存的影响Note： A. Representative microscopic images of IHC staining of APOBEC3B in ocular melanomas and nevis. APOBEC3B positive cells were stained by brown. B. The relationship between APOBEC3B expression and overall survival in UM patients was analyzed by using TCGA database. C. The relationship between APOBEC3B expression and disease-free survival in UM patients was analyzed by using TCGA database. D. Expressions of APOBEC3B in multiple tumor types versus normal tissues were analyzed by using the online database GEPIA. TPM—transcripts per million; ACC—adrenocortical carcinoma; BLCA—bladder urothelial carcinoma; BRCA—breast invasive carcinoma; CESC—cervical squamous cell carcinoma; 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; 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; OV—ovarian serous cystadenocarcinoma; PAAD—pancreatic adenocarcinoma; PCPG—pheochromocytoma and paraganglioma; PRAD—prostate adenocarcinoma; READ—rectum adenocarcinoma; SARC—sarcoma; SKCM—skin cutaneous melanoma; STAD—stomach adenocarcinoma; TGCT—testicular germ cell tumors; THCA—thyroid carcinoma; THYM—thymoma; UCEC—uterine corpus endometrial carcinoma; UCS—uterine carcinosarcoma.

Fig 1 Expression of APOBEC3B in tumors and its effect on UM patient survival

图2 APOBEC3B在肿瘤细胞中的表达情况和 APOBEC3B 敲低和敲除细胞系的构建Note： A. APOBEC3B expression in UM, CM and other cell lines detected by Western blotting. B. Expression of APOBEC3B in shAPOBEC3B and shCtrlcell line. The bar chart of its statistical analysis is shown below. C. Expression of APOBEC3B in sgAPOBEC3B and Vector cell line. The bar chart of its statistical analysis is shown below. D. APOBEC3B knockout validation of sgAPOBEC3B cell line by using genomic DNA sequencing.

Fig 2 Expression of APOBEC3B in tumor cells and construction of APOBEC3B knockdown and knockout cell lines

图3 APOBEC3B的表达对OMM2.3细胞的克隆形成能力和细胞周期的调控Note： A. Representative images of colony formation assay performed on sgAPOBEC3B and Vector OMM2.3 cell lines. B. Quantitative summary of OMM2.3 colony formation assay. C. Cell cycle analysis of shAPOBEC3B and shCtrl OMM2.3 cell lines by flow cytometry. D. Quantification of the percentages of OMM2.3 cells in each phase.

Fig 3 Clonogenic ability and cell cycle regulation of OMM2.3 cells by APOBEC3B expression

图4 OMM2.3细胞中APOBEC3B对复制应激相关通路的调控Note： A. RNA-seq of shAPOBEC3B and shCtrl cell lines. B. GSEA analysis of differentially expressed genes derived from RNA-seq of shAPOBEC3B and shCtrl OMM2.3 cell lines. NES—normalized enrichment score. C. Heat map of differentially expressed genes derived from RNA-seq of shAPOBEC3B and shCtrl OMM2.3 cell lines. D. Gene expression changes of OMM2.3 after APOBEC3B knockdown were measured by RT-qPCR. E. Representative images of p-RPA32 staining in shAPOBEC3B and shCtrl OMM2.3 cell lines. F. Representative images of colony formation assay performed on shAPOBEC3B and shCtrl OMM2.3 cell lines treated with CHK1 inhibitor (CHK1i). G. Quantitative analysis of colony formation assay. ①P=0.004, ②P=0.001, ③P=0.044, ④P=0.017, ⑤P=0.000, ⑥P=0.009, ⑦P=0.006.

Fig 4 Regulation of replication stress-related pathways by APOBEC3B in OMM2.3 cells

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APOBEC3B调控葡萄膜黑色素瘤复制应激的研究

Role of APOBEC3B in regulating replication stress of uveal melanoma

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