Knockout of CTNNB1 by CRISPR-Cas9 technology inhibits cell proliferation through the Wnt/β-catenin signaling pathway
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.
Keywordsβ-catenin Cell adhesion Cell proliferation CRISPR-Cas9 CTNNB1 Wnt/β-catenin signaling pathway
This work was supported by grants from the National Natural Science Foundation of China (grant number 81771226 and 81600987), Henan Province Natural Science Foundation (162300410214), Research Program of Foundation and Advanced Technology in Henan Province (grant number 162300410134), Key Scientific Research Program of Higher Education in Henan Province (grant number 16A180013 and 16A180014), Science and Technology Innovation Talents Support Program of Xinxiang City (grant number CXRC160003), Major Science and Technology Program of Xinxiang City (grant number ZD17008), Major Cultivation Plan of Scientific and Technological Achievements from Natural science class of Xinxiang Medical University (grant number 20172DCG-03), Doctoral scientific Research Program Foundation of Xinxiang Medical University (grant number XYBSKYZZ201523), Open Program of Henan key Laboratory of Biological psychiatry (grant number ZDSYS2015004), and USM fellowship.
Supplementary Table 1—The sequences of qPCR primers.
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Conflict of interest
The authors declare that they have no financial or commercial conflict of interest.