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Biotechnology Letters

, Volume 40, Issue 3, pp 501–508 | Cite as

Knockout of CTNNB1 by CRISPR-Cas9 technology inhibits cell proliferation through the Wnt/β-catenin signaling pathway

  • Lihong Guan
  • Shaoyi Zhu
  • Yawei Han
  • Ciqing Yang
  • Yanli Liu
  • Liang Qiao
  • Xiaoying Li
  • Han Li
  • Juntang LinEmail author
Original Research Paper

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-, 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.

Keywords

β-catenin Cell adhesion Cell proliferation CRISPR-Cas9 CTNNB1 Wnt/β-catenin signaling pathway 

Notes

Acknowledgement

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.

Supporting information

Supplementary Table 1—The sequences of qPCR primers.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no financial or commercial conflict of interest.

Supplementary material

10529_2017_2491_MOESM1_ESM.docx (14 kb)
Supplementary material 1 (DOCX 15 kb)

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Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2017

Authors and Affiliations

  • Lihong Guan
    • 1
    • 2
  • Shaoyi Zhu
    • 1
    • 2
  • Yawei Han
    • 3
  • Ciqing Yang
    • 1
    • 2
  • Yanli Liu
    • 1
    • 2
  • Liang Qiao
    • 1
    • 2
  • Xiaoying Li
    • 1
  • Han Li
    • 1
    • 4
  • Juntang Lin
    • 1
    • 2
    • 5
    Email author
  1. 1.College of Life Science and TechnologyXinxiang Medical UniversityXinxiangChina
  2. 2.Henan Key Laboratory of Medical Tissue RegenerationXinxiangChina
  3. 3.College of Food and BioengineeringZhengzhou University of Light IndustryZhengzhouChina
  4. 4.Regenerative Medicine Cluster, Advanced Medical and Dental InstituteUniversiti Sains MalaysiaPenangMalaysia
  5. 5.College of Biomedical EngineeringXinxiang Medical UniversityXinxiangChina

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