CRISPR-Cas9蛋白激酶文库筛选胰腺癌多药耐药共同靶点的价值

doi:10.3969/j.issn.1671-7171.2024.12.002

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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。

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	曾思媛
	兰斌
	王慧玲
	张超杰

关键词 ：胰腺肿瘤, 蛋白激酶类, 抗药性,多药, 基因文库

Abstract：【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.

Key words： Pancreatic Neoplasms Protein Kinases Drug Resistance,Multiple Gene Library

收稿日期: 2024-10-08

中图分类号:

R735.9

基金资助: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)

引用本文:

曾思媛, 兰斌, 王慧玲, 张超杰. CRISPR-Cas9蛋白激酶文库筛选胰腺癌多药耐药共同靶点的价值[J]. 医学临床研究, 2024, 41(12): 1828-1832.
ZENG Siyuan, LAN Bin, WANG Huiling, et al. Value of CRISPR-Cas9 Protein Kinase Libraries in Screening Common Targets for Multidrug Resistance in Pancreatic Cancer. JOURNAL OF CLINICAL RESEARCH, 2024, 41(12): 1828-1832.

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http://journal07.magtech.org.cn/yxlcyj/CN/10.3969/j.issn.1671-7171.2024.12.002 或 http://journal07.magtech.org.cn/yxlcyj/CN/Y2024/V41/I12/1828

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