靶向SF3B1突变型葡萄膜黑色素瘤的药物筛选  

Screening of drugs that selectively inhibit uveal melanoma cells with SF3B1 mutations

作　　者：罗昕 任重鲁 刘小莲 张贵明 黄思思 余乐[3] 李亦蕾[1] LUO Xin;REN Chonglu;LIU Xiaolian;ZHANG Guiming;HUANG Sisi;YU Le;LI Yilei(Department of Pharmacy,Nanfang Hospital,Southern Medical University,Guangzhou 510515,China;College of Medical Information Engineering,Guangdong Pharmaceutical University,Guangzhou 510006,China;School of Pharmaceutical Sciences,Southern Medical University,Guangzhou 510515,China)

机构地区：[1]南方医科大学南方医院药学部,广东广州510515 [2]广东药科大学医药信息工程学院,广东广州510006 [3]南方医科大学药学院,广东广州510515

出　　处：《南方医科大学学报》2021年第12期1835-1842,共8页Journal of Southern Medical University

基　　金：广州市科技计划项目(4001616289);吴阶平医学基金会科研项目(302.6750.2020-10-120);白求恩公益基金会科研项目(B-19-H-20200622);蕙兰基金科研项目(2021-HLJJ0328)。

摘　　要：目的构建SF3B1突变等位基因敲除的人葡萄膜黑色素瘤细胞模型,筛选靶向抑制SF3B1突变型葡萄膜黑色素瘤的药物。方法通过主成分分析法对来自TCGA数据库的葡萄膜黑色素瘤患者分成SF3B1野生型和突变型两组进行转录组可变剪接分析,研究SF3B1突变对可变剪接的影响。通过CRISPR-Cas9技术敲除Mel202细胞株的SF3B1突变等位基因,Sanger测序明确基因编辑的序列。MTT法和克隆形成实验检测Mel202细胞株和基因敲除mut-KO细胞株的增殖,RT-PCR琼脂糖电泳结合Sanger测序检测Mel202细胞株和基因敲除mut-KO细胞株的可变剪接事件。MTT法从上市药物库和生物活性化合物库筛选对SF3B1突变细胞具有选择性抑制活性的药物。结果选择性敲除Mel202细胞SF3B1突变等位基因促进了细胞的增殖(5.47±0.32 vs 10.17±0.27),改变了ZDHHC16和DYNLL1转录本的可变剪接。化合物库筛选结果显示13个化合物对SF3B1突变的Mel202细胞具有选择性的抑制活性(Fold change≥2),其中上市药物tetrandrine和lapatinib显示了较好的量效曲线。结论本研究为SF3B1突变的葡萄膜黑色素瘤患者提供了细胞筛选模型和潜在的个体化治疗药物。Objective To screen compounds that can selectively inhibit uveal melanoma cells with splicing factor 3B subunit 1(SF3B1)mutations in comparison with isogenic SF3B1 wild-type counterparts in a cell model of SF3B1 mutant allele knockout.Methods Principal component analysis was used to analyze transcriptome alternative splicing in TCGA cohorts of uveal melanoma with wild-type SF3B1 and SF3B1 mutations,and abnormal alternative splicing events derived from SF3B1 mutations were identified.The SF3B1 mutant allele in Mel202 cells was knocked out using CRISPR-Cas9 technology,and Sanger sequencing was used to verify the edited sequence.MTT and colony formation assays were used to assess the proliferation of Mel202 and Mut-KO cells.RT-PCR agarose electrophoresis combined with Sanger sequencing was used to determine alternative splicing events in Mel202 and Mut-KO cells.MTT assay was performed to screen the compounds that showed selective inhibitory effect against Mel202 cells with SF3B1 mutation.Results Specific knockout of SF3B1 mutant allele in Mel202 cells obviously promoted the cell proliferation and caused changes in alternative splicing of ZDHHC16 and DYNLL1 transcripts.The screening data showed that 13 compounds had selective inhibitory activity against Mel202 cells with SF3B1 mutation(Fold change≥2),and among them,tetrandrine and lapatinib showed good dose-effect curves.Conclusion This study provides a cell screening model for identification of potential individualized treatment drugs for patients with uveal melanoma with SF3B1 mutation.

关 键 词：葡萄膜黑色素瘤 剪切因子3B亚基1 突变 基因编辑 药物筛选 个体化治疗药物 

分 类 号：R965.1[医药卫生—药理学]