靶向抑制CDK12/13在高级别胶质瘤中的体外治疗效果和作用分子机制探究  

In vitro therapeutic effects and molecular mechanisms of targeted inhibition of CDK12/13 in high-grade gliomas

作　　者：梅艳青 韩雨洁 翁文筠 张蕾 唐玉杰 MEI Yanqing;HAN Yujie;WENG Wenyun;ZHANG Lei;TANG Yujie(Department of Pathophysiology,Shanghai Jiao Tong University School of Medicine,Shanghai 200025,China;Department of Histoembryology and Genetic Development,Shanghai Jiao Tong University School of Medicine,Shanghai 200025,China)

机构地区：[1]上海交通大学医学院病理生理学系,上海200025 [2]上海交通大学医学院组织胚胎学与遗传发育学系,上海200025

出　　处：《上海交通大学学报（医学版）》2023年第5期545-559,共15页Journal of Shanghai Jiao tong University:Medical Science

基　　金：国家自然科学基金面上项目(81772655,81972646);上海交通大学医学院高水平地方高校创新团队(SHSMU-ZDCX20212700)。

摘　　要：目的·筛选高级别胶质瘤(high-grade gliomas,HGGs)共有的表观转录靶向治疗新策略,并进行体外治疗效果的测试与相关分子机制的探究。方法·对HGGs中恶性程度和致死率均较高的多个胶质母细胞瘤(glioblastoma,GBM)和弥漫性内生性脑桥胶质瘤(diffuse intrinsic pontine glioma,DIPG)细胞系进行表观转录相关的靶向小分子药物库筛选和基于CRISPR-Cas9系统的功能基因组筛选,以寻找在GBM和DIPG中共同的表观转录靶向治疗新策略。然后针对筛选得到的目标表观转录调控因子,分别测试经CRISPR-Cas9方法敲除该基因以及对应的靶向小分子处理对GBM和DIPG细胞系的体外生长、细胞增殖与凋亡的影响。继而对靶向小分子处理的GBM和DIPG细胞系进行RNA-seq转录组分析,解析其抗肿瘤分子机制。基于此分析结果,通过实时荧光定量PCR(quantitative real-time PCR,RT-qPCR)、蛋白质印迹法以及流式细胞术进一步验证目标表观转录调控因子的抗肿瘤分子机制。结果·针对表观转录调控因子的靶向小分子药物库筛选和功能基因组筛选鉴定出CDK12和CDK13(CDK12/13)是GBM和DIPG共同的潜在治疗新靶点。在多个GBM和DIPG细胞系中,通过CRISPR-Cas9方法敲除CDK12能够显著降低其体外细胞活性。CDK12/13抑制剂SR-4835或THZ531也均能够通过拮抗细胞增殖和促进细胞凋亡造成这2类HGGs细胞系的体外生长受到显著抑制。SR-4835处理GBM和DIPG细胞之后的RNA-seq转录组分析结果表明,HGGs细胞中受CDK12/13抑制剂作用而表达显著下调的基因主要富集在转录调控、DNA损伤反应(DNA damage response,DDR)通路、泛素-蛋白酶体通路以及细胞周期等生物学过程。进一步通过一系列实验方法验证了在DIPG和GBM中,靶向抑制CDK12/13显著下调DDR相关基因的转录进而引起DNA损伤的累积,并诱导细胞发生G2-M细胞周期阻滞。结论·CDK12/13是GBM和DIPG这2类HGGs共同的潜在表观转录靶向治疗�Objective·To find novel and common targeting strategies for high-grade gliomas(HGGs)from the perspective of epigenetic and transcriptional modulators,test the therapeutic effect in vitro and investigate the related molecular mechanisms.Methods·Glioblastoma(GBM)and diffuse intrinsic pontine glioma(DIPG)cell lines with high malignancy and mortality in HGGs were selected for screening of targeted small molecule drug library related to epigenetic transcription and for functional genome screening based on the CRISPR-Cas9 technology.The effect of selected targeted epigenetic transcriptional modulators on growth,proliferation,and apoptosis of GBM and DIPG cell lines were then measured either by CRISPR-Cas9 knockout or treatment with targeted small molecule inhibitors of genes in vitro.Anti-tumor molecular mechanisms of the modulators in corresponding small molecule inhibitors-treated GBM and DIPG cells were explored via RNA-seq transcriptome analysis and further verified by real time quantitative PCR(RT-qPCR),Western blotting and flow cytometry.Results·Targeted small molecule drug library combined with functional genome screening for epigenetic transcriptional modulators identified CDK12/13 as the novel therapeutic targets for both GBM and DIPG.Knockout out of CDK12 by CRISPR-Cas9 in multiple GBM and DIPG cell lines significantly reduced their in vitro cellular activity.CDK12/13 inhibitors SR-4835 and THZ531 also significantly inhibited the growth of these two types of HGGs cell lines in vitro by antagonizing cell proliferation and promoting cell apoptosis.RNA-seq transcriptome analysis of GBM and DIPG cell lines after SR-4835 treatment showed that genes significantly down-regulated by CDK12/13 inhibitors in HGGs cells were mainly enriched in transcriptional regulation,DNA damage response(DDR)pathway,ubiquitin-proteasome pathway,and cell cycle.Furthermore,a series of experiments demonstrated that targeted inhibition of CDK12/13 significantly down-regulated the transcription of DDR-related genes,resulting in the accum

关 键 词：胶质母细胞瘤 弥漫性内生性脑桥胶质瘤 分子靶向治疗 抗肿瘤药物库筛选 CRISPR-Cas9 CDK12 CDK13 

分 类 号：R739.41[医药卫生—肿瘤]