基于CRISPR/Cas9技术的SRSF9基因敲除对成胶质细胞瘤生物学功能的影响  

Biological functions of glioblastoma cells with SRSF9 gene knockout based on CRISPR/Cas9 technology

作　　者：汪京京 胡亚玲 邹健 张博 穆会君 殷莹 WANG Jingjing;HU Yaling;ZOU Jian;ZHANG Bo;MU Huijun;YIN Ying(Center of Clinical Research,Wuxi People＇s Hospital of Nanjing Medical University,Wuxi Institute of Translational Medicine,Wuxi 214023,Jiangsu Province,China)

机构地区：[1]南京医科大学附属无锡人民医院临床研究中心无锡市转化医学研究所,江苏无锡214023

出　　处：《肿瘤》2018年第11期1011-1021,共11页Tumor

基　　金：国家自然科学基金资助项目(编号:81572468);无锡市医学重点人才培养项目(编号:ZDRC001);江苏省自然科学基金资助项目(编号:BK20151105);江苏省青年医学重点人才培养项目(编号:QNRC2016188)~~

摘　　要：目的:基于成簇规律间隔短回文重复序列(clustered regulatory interspaced shortpalindromicrepeats,CRISPR)/Cas9系统构建富含丝氨酸/精氨酸剪切因子9(serine-arginine-rich splicing factor 9,SRSF9)基因敲除的人脑胶质瘤U87稳定细胞株,研究SRSF9在人成胶质细胞瘤中的生物学功能。方法:利用CRISPR/Cas9系统构建3个针对SRSF9基因的单链向导RNA(single guide RNA,sgRNA),将其转入胶质瘤U87细胞后,采用蛋白质印迹法鉴定SRSF9基因敲除效率。采用有限稀释法挑选出3株SRSF9基因敲除的U87单克隆细胞株并扩增,采用CCK-8法和EdU掺入的FCM法检测细胞体外增殖能力,采用平板克隆形成实验和干细胞成球实验分别检测细胞体外克隆形成和成球的能力,采用裸鼠皮下成瘤实验检测细胞体内成瘤的能力。结果:3条sgRNA成功插入慢病毒载体中,且序列正确。经慢病毒感染效率分析及蛋白质印迹法鉴定证明,SRSF9基因敲除的U87多克隆细胞株构建成功;经蛋白质印迹法和DNA测序确认SRSF9基因敲除的U87单克隆细胞株构建成功,序列发生移码突变。SRSF9基因敲除后,U87单克隆细胞的增殖和克隆形成能力均明显降低(P值均<0.05),肿瘤干细胞成球能力也明显下降(P <0.001),而且在裸鼠体内的成瘤能力明显减弱(P <0.01)。结论:成功建立基于CRISPR/Cas9技术的SRSF9基因敲除的脑胶质瘤U87细胞株,并通过功能实验证明SRSF 9是成胶质细胞瘤的一个关键致癌基因。Objective： To construct a serine-arginine-rich splicing factor 9 （SRSF9） gene knockout model using clustered regulatory interspaced short palindromic repeat/Cas9 （CRISPR/Cas9） investigate the biological function of SRSF9 in system in human glioma U87 cell line, and to human glioblastoma cells. Methods： Three single guide RNAs （sgRNAs） targeting SRSF9 gene were constructed by CRISPR/Cas9 system, and the lentivirus carried each SRSF9-sgRNAs was infected into glioma U87 cells. The knockout efficiency of SRSF9 gene was identified by Western blotting. Three strains of U87 monoclonal cells with SRSF9 gene knockout were selected by limited dilution and amplified method. Then the cell proliferation in vitro was detected by CCK-8 assay and EdU-incorporated FCM method, the colony-formation ability in vitro was detected by colony-formation assay, the tumor sphere formation ability in vitro was detected by stem cell globulation assay, and the tumorigenic ability in vivo was tested by subcutaneous tumorigenesis assay in nude mice. Results： Three sgRNAs were successfully inserted into the lentivirus vector, and the sequence was correct. The infection efficiency analysis and Western blotting results showed that U87 polyclonal cell lines with SRSF9 gene knockout were successfully established. The U87 monoclonal cell strains with SRSF9 gene knockout and frameshift mutations were obtained and determined by Western blotting and DNA sequencing, respectively. The proliferation and colony-formation abilities of U87 monoclonal cells with SRSF9 gene knockout were significantly reduced （both P 〈 0.05）, the tumor sphere formation ability was significantly reduced （P 〈 0.001）, and the tumorigenesis in nude mice was also significantly decreased （P 〈 0.01）. Conclusion： The U87 cell strains with SRSF9 gene knockout are successfully established by CRISPR/Cas9 technology, and SRSF9 is proved to be a key oncogene in gliobiastoma.

关 键 词：神经胶质瘤 基因敲除技术 细胞增殖 基因 SRSF9 CRISPR/Cas9技术 

分 类 号：R730.264[医药卫生—肿瘤]