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。

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.