Effect of deletion of G protein-coupled receptor 107 on the biological behaviour of HaCaT cells
10.19405/j.cnki.issn1000-1492.2025.03.001
- Author:
Jing Wang
1
;
Wei Zhao
1
;
Deping Xu
1
;
Kainan Liao
1
;
Dandan Zang
2
;
Haisheng Zhou
3
Author Information
1. Dept of Biochemistry， Anhui Medical University，Hefei 230032
2. Center Scientific Research，Anhui Medical University，Hefei 230032
3. Dept of Biochemistry， Anhui Medical University，Hefei 230032; Center Scientific Research，Anhui Medical University，Hefei 230032
- Publication Type:Journal Article
- Keywords:
G protein-coupled receptor 107;
HaCaT cells;
CRISPR / Cas9;
cell proliferation;
cell cycle
- From:
Acta Universitatis Medicinalis Anhui
2025;60(3):385-391
- CountryChina
- Language:Chinese
-
Abstract:
Objective :To construct a human keratinocyte-forming cell line(HaCaT) with stable knockout of the G protein-coupled receptor 107(GPR107) gene, and to preliminarily investigate the effect of GPR107 deletion on the biological behaviour of HaCaT cells.
Methods :Using CRISPR/Cas9 gene editing technology, HaCaT cells with knockout ofGPR107gene were constructed and monoclonal cells with GPR107 deletion were obtained by limited dilution method. Genomic DNA was amplified using Western blot and PCR and sequenced to validate the single-cell clones with knockdown of GPR107. The cell cycle changes were detected by flow cytometry; cell proliferation was detected by CCK-8; apoptosis was detected by flow cytometry; changes in cell differentiation markers were detected by Western blot; cell migration ability was analyzed by cell scratch assay and other methods.
Results :LentiCas9-Blast and plenti-guide-RNA-GPR107 plasmids were successfully transfected into HaCaT cells, 21 monoclonal cell lines were obtained by limited dilution, and Western blot showed that the GPR107 expression was significantly reduced in 8 of them; PCR sequencing of the cellular genome was used, which resulted in the obtainment of C4 and 2D8GPR107-/-HaCaT monoclonal cell lines. CCK-8 assay and flow cytometry assay showed thatGPR107gene deletion resulted in G0G1phase block, significantly weakened proliferation ability and increased apoptosis level of HaCaT cells. Western blot found that the differentiation of HaCaT cells accelerated after knockdown ofGPR107. Additionally the results of the cell scratch assay indicated that the migration ability of HaCaT cells was enhanced after knockdown ofGPR107. The results showed that the migration ability of HaCaT cells was enhanced after knockdown ofGPR107.
Conclusion :HaCaT cell line withGPR107gene deletion is successfully constructed, GPR107 deletion blocks the G0G1phase of HaCaT cells, which inhibiting the proliferation of HaCaT cells and promoted apoptosis, and it was found that the differentiation and migration of HaCaT cells were enhanced after knocking downGPR107.
- Full text:2026012216570193645G蛋白偶联受体107的缺失...CaT细胞生物学行为的影响_汪璟.pdf
