利用CRISPR/Cas9筛选膀胱癌T24细胞生长必需基因  

A genome-wide CRISPR/Cas9 screen in bladder cancer T24 cells identifies essential growth genes

作　　者：韦连心 陶玉婷[2] 黄浩轩[2] 冯超 陆钦晨 李天宇[2,3] 王秋雁[1,2] Wei Lianxin;Tao Yuting;Huang Haoxuan;Feng Chao;Lu Qinchen;Li Tianyu;Wang Qiuyan(Department of Biochemistry and Molecular Biology,Basic Medical College,Nanning 530021,China;Center For Genomic and Personalized Medicine,Guangxi Medical University,Nanning 530021,China;The First Affiliated Hospital of Guangxi Medical University,Nanning 530021,China)

机构地区：[1]广西医科大学基础医学院生物化学与分子生物学教研室,南宁530021 [2]广西医科大学基因组与个体化医学研究中心,南宁530021 [3]广西医科大学第一附属医院泌尿外科,南宁530021

出　　处：《广西医科大学学报》2020年第12期2117-2122,共6页Journal of Guangxi Medical University

基　　金：国家自然科学基金项目(No.81760454);广西自然科学基金项目(No.2016GXNSFGA380006,No.2017GXNSFAA198323,No.2019GXNSFAA245097);广西科技开发课题资助项目(No.桂科AD17195090)。

摘　　要：目的:利用CRISPR/Cas9基因编辑技术,筛选维持膀胱癌T24细胞正常生长必需基因,为膀胱癌靶向治疗提供新的思路。方法:将人全基因组文库质粒进行扩繁并包装慢病毒,然后感染T24野生型细胞,用嘌呤霉素(puromycin)进行筛选。为了获取T24细胞生长必需基因表达变化情况,分别收集第1天的活细胞作为对照组,培养第14天的细胞作为实验组,分别提取两组细胞DNA,进行全基因组测序和生物信息学分析获得候选基因。对候选基因进行功能注释和通路分析,并结合TCGA数据库进行生存分析,了解这些基因的主要富集通路及对预后的影响。结果:筛选出了971个可能维持膀胱癌细胞正常生长的必需基因,这些基因主要富集在脂质代谢、糖代谢、细胞周期和蛋白质合成等通路中,其中部分基因可能影响膀胱癌预后。结论:本研究对于进一步验证这些基因在膀胱癌T24细胞中的功能和机制具有重要意义。Objective:CRISPR/Cas9 gene editing technology was adaptedto screen the genes essential for T24 cells growth,and provide a new idea for targeted therapy of bladder cancer.Methods:The whole human genome library plasmid was amplified and packaged with lentivirus,infected with T24 wild-type cells and screened with puromycin.In order to obtain the expression changes of essential genes for the growth of T24 cells,the living cells on the 1st day were collected as the control group,and the cultured cells on the 14th day were collected as the experimental group.The cells were extracted from two groups,and whole genome sequencing and bioinformatics analysis were performed in order to select the candidate genes.Performed functional annotation and pathway analysis of candidate genes,and conducted survival analysis combined with TCGA database to understand the main enrichment pathways of these genes and their impact on prognosis.Results:A total of 971 genes that might be necessary to maintain the normal growth of bladder cancer cells werescreened out.These geneswere concentrated in the pathways of lipid metabolism,glucose metabolism,cell cycle,and protein synthesis,and some of them may affect the prognosis of bladder cancer.Conclusion:This research is important for further validation on the function and mechanism of these genes in bladder cancer T24 cells.

关 键 词：生长必需基因 CRISPR/Cas9 膀胱癌 

分 类 号：R730.261[医药卫生—肿瘤]