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CRISPR-Cas9-Mediated NPC1 Gene Deletion Enhances HEK 293 T Cell Adhesion by Regulating E-Cadherin

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Abstract

NPC1 gene encodes a transmembrane glycoprotein on the late endosome/lysosomal membrane. Its mutation leads to a rare and aggravated autosomal recessive neurovisceral condition, termed Niemann-Pick disease type C1 (NPC1), which is characterized by progressive neurodegeneration, visceral symptoms, and premature death. To investigate the influence of NPC1 gene deletion on cell morphology, adhesion, proliferation, and apoptosis, CRISPR-Cas9 technology was used to knockout the NPC1 gene in HEK 293 T cells. Sanger sequencing, western blotting, and immunofluorescence were used to confirm successful NPC1 ablation. Filipin staining results indicated that deletion of NPC1 gene led to accumulation of unesterified cholesterol in HEK 293 T cells. Phalloidin staining results revealed cell aggregation, synapse shortening, nuclear enlargement, and cytoskeleton filamentous actin thinning in HEK 293 T cells with NPC1 gene mutation. Furthermore, NPC1 gene mutated HEK 293 T cell showed enhanced cell adhesion, inhibited cell proliferation, and increased cell apoptosis. In addition, NPC1 gene mutations significantly increased the protein expression levels of E-cadherin and γ-catenin and significantly decreased the protein expression levels of Wnt 3a, c-Myc, and cyclin D1. These results suggest that NPC1 may regulate cell adhesion by affecting the cadherin–catenin complex through E-cadherin, and that the classical Wnt signaling pathway may be inhibited by restricting β-catenin from entering the nucleus to inhibit cell proliferation.

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Funding

This work was supported by grants from the National Natural Science Foundation of China (Grant Nos. 81801127, 81771226, 81800792, and U1804186), Science and Technology Innovative Research Team in Higher Educational Institutions of Henan Province (Grant No. 19IRTSTHN003), Henan Province Science and Technology Project (Grant No. 212102310215), Natural Science Foundation of Henan Province (Grant No. 212300410225), Foundation for the University Key Teacher by the Henan Province (Grant No. 2020GGJS148), Major Cultivation Plan of Scientific and Technological Achievements from Natural science class of Xinxiang Medical University (Grant No. 20172DCG-03), Doctoral Scientific Research Program Foundation of Xinxiang Medical University (Grant No. XYBSKYZZ201523), and Open Program of Henan Key Lab of Biological Psychiatry (Grant No. ZDSYS2015004), Preferential Scientific Research Foundation for Overseas Scholars of Henan Province (Grant No. 40), Research Innovation Support Program for Postgraduates of Xinxiang Medical University (Grant No. YJSCX202135Y).

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Authors and Affiliations

  1. School of Life Sciences and Technology, Xinxiang Medical University, Xinxiang, China

    Zisen Jia, Minlin Yang, Yanchun Zhao, Xiaoying Li, Ciqing Yang, Liang Qiao, Han Li, Juntang Lin & Lihong Guan

  2. Henan Key Laboratory of Medical Tissue Regeneration, Xinxiang, China

    Zisen Jia, Minlin Yang, Yanchun Zhao, Xiaoying Li, Ciqing Yang, Liang Qiao, Han Li, Jiang Du, Juntang Lin & Lihong Guan

  3. Henan Joint International Research Laboratory of Stem Cell Medicine, Stem Cell and Biotherapy Engineering Research Center of Henan, Xinxiang, China

    Zisen Jia, Minlin Yang, Yanchun Zhao, Xiaoying Li, Ciqing Yang, Liang Qiao, Han Li, Jiang Du, Juntang Lin & Lihong Guan

  4. School of Medical Engineering, Xinxiang Medical University, Xinxiang, China

    Jiang Du & Juntang Lin

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  1. Zisen Jia
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  2. Minlin Yang
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  3. Yanchun Zhao
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  4. Xiaoying Li
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  6. Liang Qiao
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  7. Han Li
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  8. Jiang Du
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  9. Juntang Lin
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  10. Lihong Guan
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Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by ZJ, MY and YZ. The first draft of the manuscript was written by ZJ and LG, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Correspondence to Juntang Lin or Lihong Guan.

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Jia, Z., Yang, M., Zhao, Y. et al. CRISPR-Cas9-Mediated NPC1 Gene Deletion Enhances HEK 293 T Cell Adhesion by Regulating E-Cadherin. Mol Biotechnol 65, 252–262 (2023). https://doi.org/10.1007/s12033-022-00503-2

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  • DOI: https://doi.org/10.1007/s12033-022-00503-2

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