CRISPR-Cas9蛋白激酶文库筛选胰腺癌多药耐药共同靶点的价值  

Value of CRISPR-Cas9 Protein Kinase Libraries in Screening Common Targets for Multidrug Resistance in Pancreatic Cancer

作　　者：曾思媛 兰斌 王慧玲[1] 张超杰[1] ZENG Siyuan;LAN Bin;WANG Huiling(Hunan Provincial People's Hospital(The First Affiliated Hospital of Hunan Normal University),Changsha Hunan 410005)

机构地区：[1]湖南省人民医院(湖南师范大学附属第一医院),湖南长沙410005

出　　处：《医学临床研究》2024年第12期1828-1832,共5页Journal of Clinical Research

基　　金：国家自然科学基金(82403714);湖南省自然科学基金(2023JJ40375);湖南省卫生健康委员会科研课题(W20243025)。

摘　　要：【目的】分析CRISPR-Cas9蛋白激酶文库筛选胰腺癌(PDAC)多药耐药共同靶点的价值。【方法】在KPC小鼠模型衍生的PDAC细胞系TB32047中通过慢病毒转染全蛋白激酶组文库,分别使用IC90药物浓度的奥沙利铂、氟尿嘧啶、伊利替康连续处理上述细胞3周。通过CRISPR-Cas9功能缺失筛选,进行下一代测序和MAGeCK VISPR分析以鉴定候选基因。采用MTT、流式细胞术、细胞集落形成实验检测细胞周期蛋白依赖性激酶7(CDK7)耗竭或靶向抑制与PDAC FOLFIRINOX化疗方案的协同作用。【结果】CDK7在三种化疗药物筛选中均排名靠前,是PDAC多药耐药的共同靶点。利用CRISPR-Cas9完全敲除CDK7或使用THZ1抑制CDK7的表达后,PDAC细胞增殖减慢,细胞周期阻滞,细胞凋亡增加,细胞集落形成能力减弱,并与FOLFIRINOX协同促进PDAC细胞凋亡。【结论】CDK7为PDAC多药耐药的共同靶点,CDK7是抑制PDAC化疗耐药性的新靶点。【Objective】To analyze the value of CRISPR-Cas9 protein kinase library in screening common targets for multi-drug resistance in pancreatic cancer.【Methods】PDAC cell line TB32047 derived from KPC mouse model was transfected with lentivirus and treated with oxaliplatin,fluorouracil and ilictecan at IC90 concentration for 3 weeks.Next generation sequencing and MAGeCK VISPR analysis were performed to identify candidate genes through CRISPR-Cas9 function loss screening.The synergistic effect of cyclin-dependent kinase 7(CDK7)depletion or targeted inhibition with FOLFIRINOX chemotherapy for pancreatic cancer was examined by MTT,flow cytometry,and colony formation assay.【Results】CDK7 ranked high in the screening of three chemotherapy drugs,and was a common target of multi-drug resistance in pancreatic cancer.After completely knocking out CDK7 with CRISPR-Cas9 or inhibiting CDK7 expression with THZ1,pancreatic cancer cell proliferation was slowed down,the cell cycle was blocked and apoptosis increased,while colony-forming ability decreased,and together with FOLFIRINOX,they promoted apoptosis of pancreatic cancer cells.【Conclusion】CDK7 is a common target of multi-drug resistance in pancreatic cancer,and CDK7 is a new target to overcome chemotherapy resistance in pancreatic cancer.

关 键 词：胰腺肿瘤 蛋白激酶类 抗药性 多药 基因文库 

分 类 号：R735.9[医药卫生—肿瘤]