利用CRISPR/Cpf1系统构建稳定敲除LncRNA458314的肾癌细胞株及其功能探讨  

Construction of a stable LncRNA458314 knockout renal cancer cell line using the CRISPR/Cpf1 system and its function evaluation

作　　者：周洋 陈兵海[1] 王诚悦 ZHOU Yang;CHEN Bing-hai;WANG Cheng-yue(Department of Urology, Affiliated Hospital of Jiangsu University, Zhenjiang Jiangsu 212001, China)

机构地区：[1]江苏大学附属医院泌尿外科

出　　处：《江苏大学学报（医学版）》2020年第1期29-33,共5页Journal of Jiangsu University：Medicine Edition

摘　　要：目的:通过新基因编辑系统CRISPR/Cpf1稳定敲除肾癌细胞769P中的LncRNA458314,并初步研究LncRNA458314在肾癌中的作用。方法:使用CRISPR/Cpf1系统敲除肾癌细胞769P的LncRNA458314,并通过荧光定量PCR检测敲除效率。通过CCK8实验检测LncRNA458314敲除后769P细胞的增殖能力;蛋白质印迹法检测LncRNA458314敲除后769P细胞pAKT、pERK和LC3A/B蛋白的表达。结果:荧光定量PCR结果表明CRISPR/Cpf1系统成功敲除了目标LncRNA458314,并且通过多次荧光定量PCR检测对比后,挑选了两个稳定LncRNA458314低表达的769P单克隆细胞(6#和13#)用于后续实验。CCK8实验表明,LncRNA458314下调后,肾癌769P细胞的增殖能力明显下降;蛋白质印迹结果表明,LncRNA458314敲除后的769P细胞pAKT、pERK表达水平明显下降,LC3A/B表达水平明显上升。结论:LncRNA458314可能通过调控pAKT、pERK和LC3A/B表达发挥其生物学功能。Objective:A new gene editing system,CRISPR/Cpf1,was used to stably knock out LncRNA458314 in renal cell carcinoma 769P,and to preliminarily study the role of LncRNA458314 in renal cancer.Methods:LncRNA458314 of renal cell carcinoma 769P was knocked out using the CRISPR/Cpf1 system,and the knockout efficiency was evaluated by fluorescent quantitative PCR.CCK8 assay was used to detect the proliferation of 769P cells after LncRNA458314 knockout;Western blotting was used to detect the expression of pAKT,pERK and LC3A/B proteins in 769P cells after LncRNA458314 knockout.Results:The quantitative PCR results showed that the CRISPR/Cpf1 system successfully knocked out the target LncRNA458314,and after multiple fluorescent quantitative PCR detection and comparison,two 769P/LncRNA458314 monoclonal cells 6#and 13#with stable low expression of LncRNA458314 were selected for subsequent experiment.CCK8 experiments showed that after LncRNA458314 was knocked out,the proliferative capacity of renal cancer 769P cells was significantly reduced;Western blotting results showed that the expression levels of pAKT and pERK in 769P cells after knockout of LncRNA458314 were significantly reduced,and LC3A/B expression levels were significantly increased.Conclusion:LncRNA458314 may exert its biological functions by regulating the expression of pAKT,pERK and LC3A/B.

关 键 词：肾癌 长链非编码RNA CRISPR/Cpf1 LncRNA458314 基因编辑系统 

分 类 号：R737.11[医药卫生—肿瘤]