利用CRISPR/Cas9 sgRNA文库筛选调控结直肠癌对奥沙利铂敏感性的表观遗传相关基因  

Screening for Epigenetic Related Genes Regulating the Sensitivity of Colorectal Cancer to Oxaliplatin Using an CRISPR/Cas9 sgRNA Library

作　　者：付亚坤 贾林创 穆标[1] FU Ya-Kun;JIA Lin-Chuang;MU Biao(NHC Key Laboratory of Hormones and Development,Tianjin Key Laboratory of Metabolic Diseases,Chu Hsien-I Memorial Hospital&Tianjin Institute of Endocrinology,Tianjin Medical University,Tianjin 300134,China;Department of Pathophysiology,School of Basic Medical Sciences,Tianjin Medical University,Heping,Tianjin 300070,China)

机构地区：[1]天津医科大学朱宪彝纪念医院,天津市内分泌研究所,国家卫健委激素与发育重点实验室,天津市代谢性疾病重点实验室,天津300134 [2]天津医科大学基础医学院生理学与病理生理学系,天津300070

出　　处：《中国生物化学与分子生物学报》2024年第12期1698-1708,共11页Chinese Journal of Biochemistry and Molecular Biology

基　　金：天津市医学重点学科(专科)建设项目(No.TJYXZDXK-032A);天津医科大学朱宪彝纪念医院科研基金(No.ZXY-YJJ2021-1)资助。

摘　　要：结直肠癌是最常见的消化系统恶性肿瘤之一,以奥沙利铂(oxaliplatin,OXA)为基础的联合化疗方案是临床上治疗中晚期病人最常用的策略,但耐药性的产生极大限制了化疗的效果,是导致化疗失败的主要原因。因耐药发生机制未明,亟需一种高通量、高特异性的测序方法探索奥沙利铂耐药性产生的原因。CRISPR/Cas9(clustered regularly interspaced short palindromic repeats/Cas9)文库筛选是近年快速发展起来高通量筛选肿瘤耐药基因的新技术,但尚未在结直肠癌对奥沙利铂耐药性产生的基因筛选中应用。我们构建了含有5256个小向导RNA(small-guide RNA,sgRNA)的针对910个人类表观遗传相关基因的sgRNA文库,利用慢病毒包装,并通过蛋白质免疫印迹检测,以及流式细胞术检测确定病毒感染的条件,将慢病毒感染复数(multiplicity of infection,MOI)维持在30%以下,使其保证每1个细胞感染1条sgRNA并敲除1个基因;使用慢病毒携带文库感染结直肠癌细胞HCT116和SW620,通过阳性筛选策略获得单克隆并进行扩增,通过测序筛选得到21个调控结直肠癌细胞对奥沙利铂敏感的基因,通过分别敲除候选基因发现,敲除TDRKH、ALKBH3、UNKL、TTF2、TNKS、AURKA、RBM12、ELAVL2、LSM5、NOL8、PRPF 3基因可以显著提高奥沙利铂对结直肠癌细胞的半数致死量(IC_(50))(P<0.05),其中ALKBH3、AURKA和RBM 12等3个基因的高表达与临床的预后具有显著的相关性(总生存期:P=0.043,P<0.0001,P=0.045;无复发生存期:P=0.004,P=0.0019,P=0.0064)。研究证明,CRISPR/Cas9文库是筛选肿瘤敏感性基因的高通量方法,可为进一步探究结直肠癌对奥沙利铂敏感性的机制提供靶点参考。Colorectal cancer is one of the most common malignant tumors of the digestive system.Oxaliplatin(OXA)-based combination chemotherapy is the most commonly used strategy for treating patients with advanced-stage disease in clinical practice.However,the development of resistance greatly limits the effectiveness of chemotherapy and is a major cause of treatment failure.Due to the unknown mechanisms of resistance,there is an urgent need for a high-throughput,highly specific sequencing method to explore the causes of oxaliplatin resistance.Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9(CRISPR/Cas9)is a rapidly advancing high-throughput technology that can be employed for screening resistance genes.However,its role in identifying genes involved in oxaliplatin resistance in colorectal cancer remains unclear.We constructed an sgRNA library containing 5256 small-guide RNAs(sgRNAs)targeting 910 human epigenetic-related genes,using lentivirus packaging.By determining the viral infection conditions through protein immunoblotting and flow cytometry,we maintained the multiplicity of infection(MOI)below 30%to ensure that each cell is infected with only one sgRNA,thereby knocking out one gene.Colorectal cancer cells HCT116 and SW620 were infected with lentivirus carrying library,and single clones were obtained and expanded through a positive selection strategy.By the positive selection strategy,we identified 21 genes that regulate the sensitivity of colorectal cancer cells to oxaliplatin.By knocking out of candidate genes,we observed that deletion of TDRKH,ALKBH3,UNKL,TTF2,TNKS,AURKA,RBM12,ELAVL2,DKC1,LSM5,NOL 8 and PRPF 3 significantly increased the half-maximal inhibitory concentration(IC_(50))of oxaliplatin in colorectal cancer cells(P<0.05).Among them,high expression of the ALKBH3,AURKA,and RBM 12 genes was significantly correlated with clinical prognosis(overall survival:P=0.043,P<0.0001,P=0.045;recurrence-free survival:P=0.004,P=0.0019,P=0.0064).Our study demonstrates that the CRISPR/C

关 键 词：结直肠癌 奥沙利铂 肿瘤耐药性 CRISPR/Cas9文库筛选 

分 类 号：Q78[生物学—分子生物学]