Lihong Guan1,2, Shaoyi Zhu1,2, Yawei Han3, Ciqing Yang1,2, Yanli Liu1,2, Liang Qiao1,2, Xiaoying Li1, Han Li1,4, Juntang Lin5,6,7. 1. College of Life Science and Technology, Xinxiang Medical University, Xinxiang, China. 2. Henan Key Laboratory of Medical Tissue Regeneration, Xinxiang, China. 3. College of Food and Bioengineering, Zhengzhou University of Light Industry, Zhengzhou, China. 4. Regenerative Medicine Cluster, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Penang, Malaysia. 5. College of Life Science and Technology, Xinxiang Medical University, Xinxiang, China. linjtlin@126.com. 6. Henan Key Laboratory of Medical Tissue Regeneration, Xinxiang, China. linjtlin@126.com. 7. College of Biomedical Engineering, Xinxiang Medical University, Xinxiang, China. linjtlin@126.com.
Abstract
OBJECTIVE: To study the effects of CTNNB1 gene knockout by CRISPR-Cas9 technology on cell adhesion, proliferation, apoptosis, and Wnt/β-catenin signaling pathway. RESULTS: 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). CONCLUSIONS: Knockout of CTNNB1 disturbed Wnt/β-catenin signaling pathway and significantly inhibited adhesion and proliferation of HEK 293T cells.
OBJECTIVE: To study the effects of CTNNB1 gene knockout by CRISPR-Cas9 technology on cell adhesion, proliferation, apoptosis, and Wnt/β-catenin signaling pathway. RESULTS: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). CONCLUSIONS: Knockout of CTNNB1 disturbed Wnt/β-catenin signaling pathway and significantly inhibited adhesion and proliferation of HEK 293T cells.