Screening for Epigenetic Related Genes Regulating the Sensitivity of Colorectal Cancer to Oxaliplatin Using an CRISPR/Cas9 sgRNA Library
10.13865/j.cnki.cjbmb2024.11.1307
- VernacularTitle: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
- Author:
Ya-Kun FU
1
;
Lin-Chuang JIA
;
Biao MU
Author Information
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
- Publication Type:Journal Article
- Keywords:
colorectal cancer;
oxaliplatin;
chemoresistance;
CRISPR/Cas9 library screenin
- From:
Chinese Journal of Biochemistry and Molecular Biology
2024;40(12):1698-1708
- CountryChina
- Language:Chinese
-
Abstract:
Colorectal cancer is one of the most common malignant tumors of the digestive system.Oxali-platin(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 mecha-nisms 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 de-termining 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,NOL8 and PRPF3 significantly in-creased the half-maximal inhibitory concentration (IC50) of oxaliplatin in colorectal cancer cells (P<0.05) .Among them,high expression of the ALKBH3,AURKA,and RBM12 genes was significantly cor-related with clinical prognosis (overall survival:P=0.043,P<0.0001,P=0.045;recurrence-free sur-vival:P=0.004,P=0.0019,P=0.0064) .Our study demonstrates that the CRISPR/Cas9 library is a high-throughput method for screening tumor sensitivity genes,providing target references for further ex-ploring the mechanism of colorectal cancer sensitivity to oxaliplatin.
