关键词: 胰腺癌, XPO1, CRISPR/Cas9, 基因敲除

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