利用CRISPR/Cas9技术敲除XPO1基因对胰腺癌细胞株生物学功能的影响  

Effect on biological function after XPO1 knockout in pancreatic cancer cell line via CRISPR/Cas9

作　　者：黄娴娴 李苑华 黄凤婷[1] 张世能[1] 庄燕妍[1] HUANG Xian-xian;LI Yuan-hua;HUANG Feng-ting;ZHANG Shi-neng;ZHUANG Yan-yan(Department of Gastroenterology,Sun Yat Sen Memorial Hospital,Sun Yat-sen University,Guangzhou 510120,China)

机构地区：[1]中山大学孙逸仙纪念医院消化内科,广州510120

出　　处：《岭南现代临床外科》2020年第3期280-284,290,共6页Lingnan Modern Clinics in Surgery

基　　金：国家自然科学基金(81572348);广东省科技计划项目(2016A020215060)。

摘　　要：目的利用CRISPR/Cas9技术构建XPO1基因敲除的胰腺癌细胞株MIA-Paca2,初步探讨XPO1敲除对细胞生物学功能的影响。方法构建sgRNA-XPO1质粒,包装成慢病毒感染MIA-Paca2细胞,用合适浓度的嘌呤霉素筛选获得XPO1敲除的稳转株,DNA测序及蛋白质印迹法检测XPO1敲除效率;流式细胞分析及CCK8实验分别检测细胞凋亡、细胞周期及其对吉西他滨的半数抑制浓度(IC_(50))。结果 DNA测序显示编码XPO1蛋白的基因序列发生移码突变,蛋白质印迹法、流式细胞技术及CCK8结果显示XPO1基因敲除后的MIA-Paca2细胞株XPO1蛋白表达量明显降低,细胞凋亡增多,G2/M期细胞比例增加,对吉西他滨IC50降低。结论利用CRISPR/Cas9成功构建XPO1基因敲除的MIA-Paca2细胞株,XPO1基因敲除可促进细胞凋亡,引起细胞周期G2/M期阻滞,增强吉西他滨化疗敏感性。Objective To construct an XPO1-knockout pancreatic cancer cell line MIA-Paca2 using CRISPR/Cas9 technology,and to investigatethe effect of XPO1-knockout on cell biological function.Methods The sgRNA-XPO1 plasmid was constructed,packaged into lentivirus-infected MIA-Paca2 cells,and stable transgenic strains of XPO1 knockout were obtained by screening with a suitable concentration of puromycin.DNA sequencing and Western blotting were used to detect the efficiency of XPO1 knock-out;Flow cytometry analysis and CCK8 assay were used to detect apoptosis,cell cycle and thehalf inhibitory concentration(IC50)of gemcitabine.Results DNA sequencing showed a frameshift mutation in the gene sequence encoding XPO1 protein.Western blotting showed that the expression of XPO1 protein in the MIA-Paca2 cell line after XPO1 gene knockout was significantly reduced.Flow cytometry analysis and CCK8 assay suggested that apoptosis and the proportion of G2/M cells increased after XPO1 gene knock-out,while IC50 on gemcitabine decreased.Conclusion XPO1-knockout MIA-Paca2 was successfully constructed via CRISPR/Cas9.XPO1 knockout can promote apoptosis,induce G2/M phase arrest in the cell cycle,and enhance the chemotherapy sensitivity to gemcitabine.

关 键 词：CRISPR/Cas9 XPO1 基因敲除 胰腺癌 

分 类 号：R735.9[医药卫生—肿瘤]