Journal of Shanghai Jiao Tong University (Medical Science) ›› 2022, Vol. 42 ›› Issue (8): 1034-1044.doi: 10.3969/j.issn.1674-8115.2022.08.008

• Basic research • Previous Articles    

Role of APOBEC3B in regulating replication stress of uveal melanoma

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

  1. 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;
  • 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)


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

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