Abstract: 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 knockout; 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 knockout, 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.

Key words: clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 （CRISPR/Cas9）, XPO1, pancreatic cancer, Gene knockout

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（IC₅₀）。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。

关键词: 胰腺癌, XPO1, CRISPR/Cas9, 基因敲除