Guan Lihong, Zhu Shaoyi, Han Yawei, Yang Ciqing, Liu Yanli, Qiao Liang, Li Xiaoying, Li Han, Lin Juntang
College of Life Science and Technology, Xinxiang Medical University, Xinxiang, China.
Henan Key Laboratory of Medical Tissue Regeneration, Xinxiang, China.
Biotechnol Lett. 2018 Mar;40(3):501-508. doi: 10.1007/s10529-017-2491-2. Epub 2017 Dec 16.
To study the effects of CTNNB1 gene knockout by CRISPR-Cas9 technology on cell adhesion, proliferation, apoptosis, and Wnt/β-catenin signaling pathway.
CTNNB1 gene of HEK 293T cells was knocked out by CRISPR-Cas9. This was confirmed by sequencing and western blotting. Methylthiazolyl-tetrazolium bromide assays indicated that deletion of β-catenin significantly weakened adhesion ability and inhibited proliferation rate (P < 0.01) of HEK 293T cells. Nevertheless, deletion of β-catenin did not affect apoptosis of HEK 293T cells, which was analyzed by flow cytometry with Annexin V-fluorescein isothiocyanate/propidium iodide double staining. In addition, expression level of GSK-3β, CCND1, and CCNE1 detected by qPCR and expression level of N-Cadherin and cyclin D1 detected by western blotting were significantly decreased (P < 0.01) while expression of γ-catenin detected by western blotting was significantly increased (P < 0.001).
Knockout of CTNNB1 disturbed Wnt/β-catenin signaling pathway and significantly inhibited adhesion and proliferation of HEK 293T cells.
研究利用CRISPR-Cas9技术敲除CTNNB1基因对细胞黏附、增殖、凋亡及Wnt/β-连环蛋白信号通路的影响。
采用CRISPR-Cas9技术敲除HEK 293T细胞的CTNNB1基因，测序及蛋白质免疫印迹法证实敲除成功。噻唑蓝检测显示，β-连环蛋白缺失显著削弱HEK 293T细胞的黏附能力并抑制其增殖率（P<0.01）。然而，利用膜联蛋白V-异硫氰酸荧光素/碘化丙啶双染法通过流式细胞术分析发现，β-连环蛋白缺失不影响HEK 293T细胞的凋亡。此外，实时定量聚合酶链反应检测的GSK-3β、CCND1和CCNE1表达水平以及蛋白质免疫印迹法检测的N-钙黏蛋白和细胞周期蛋白D1表达水平均显著降低（P<0.01），而蛋白质免疫印迹法检测的γ-连环蛋白表达显著升高（P<0.001）。
敲除CTNNB1基因会扰乱Wnt/β-连环蛋白信号通路，并显著抑制HEK 293T细胞的黏附与增殖。
