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. 2023 Feb 7;14(2):423.
doi: 10.3390/genes14020423.

Effect of CUX1 on the Proliferation of Hu Sheep Dermal Papilla Cells and on the Wnt/β-Catenin Signaling Pathway

Hui Zhou  1   2 , Sainan Huang  1   2 , Xiaoyang Lv  2   3 , Shanhe Wang  1   2 , Xiukai Cao  2   3 , Zehu Yuan  2   3 , Tesfaye Getachew  4 , Joram M Mwacharo  4 , Aynalem Haile  4 , Kai Quan  5 , Yutao Li  6 , Antonio Reverter  6 , Wei Sun  1   2   3   7
Affiliations

Effect of CUX1 on the Proliferation of Hu Sheep Dermal Papilla Cells and on the Wnt/β-Catenin Signaling Pathway

Hui Zhou et al. Genes (Basel). .

Abstract

CUT-like homeobox 1 protein (CUX1), also called CUX, CUTL1, and CDP, is a member of the DNA-binding protein homology family. Studies have shown that CUX1 is a transcription factor that plays an important role in the growth and development of hair follicles. The aim of this study was to investigate the effect of CUX1 on the proliferation of Hu sheep dermal papilla cells (DPCs) to reveal the role of CUX1 in hair follicle growth and development. First, the coding sequence (CDS) of CUX1 was amplified by PCR, and then CUX1 was overexpressed and knocked down in DPCs. A Cell Counting Kit-8 (CCK8), 5-ethynyl-2-deoxyuridine (EdU), and cell cycle assays were used to detect the changes in the proliferation and cell cycle of DPCs. Finally, the effects of overexpression and knockdown of CUX1 in DPCs on the expression of WNT10, MMP7, C-JUN, and other key genes in the Wnt/β-catenin signaling pathway were detected by RT-qPCR. The results showed that the 2034-bp CDS of CUX1 was successfully amplified. Overexpression of CUX1 enhanced the proliferative state of DPCs, significantly increased the number of S-phase cells, and decreased the number of G0/G1-phase cells (p < 0.05). CUX1 knockdown had the opposite effects. It was found that the expression of MMP7, CCND1 (both p < 0.05), PPARD, and FOSL1 (both p < 0.01) increased significantly after overexpression of CUX1 in DPCs, while the expression of CTNNB1 (p < 0.05), C-JUN, PPARD, CCND1, and FOSL1 (all p < 0.01) decreased significantly. In conclusion, CUX1 promotes proliferation of DPCs and affects the expression of key genes of the Wnt/β-catenin signaling pathway. The present study provides a theoretical basis to elucidate the mechanism underlying hair follicle development and lambskin curl pattern formation in Hu sheep.

Keywords: CUT-like homeobox 1 protein; Hu sheep; Wnt/β-catenin; dermal papilla cells.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The relative expression level of CUX1 mRNA in different tissues of Hu sheep.
Figure 2
Figure 2
mRNA and protein expression levels of CUX1 in Hu sheep DPCs after CUX1 overexpression. (A)The relative expression levels of CUX1 mRNA. (B)The relative expression levels of CUX1 protein. (C) The relative expression levels of CUX1 protein were analyzed by Image Lab. (*) stands for p < 0.05, (**) stands for p < 0.01.
Figure 3
Figure 3
CUX1 mRNA and protein expression levels in Hu sheep DPCs after knockdown by siRNA-232. (A) The relative expression levels of CUX1 mRNA. (B) The relative expression levels of CUX1 protein. (C) The relative expression levels of CUX1 protein were analyzed using Image Lab. * p < 0.05, ** p < 0.01.
Figure 4
Figure 4
Overexpression of CUX1 promoted proliferation of Hu sheep DPCs. (A) mRNA expression levels of PCNA, CYCLIN D1, and CDK2 after CUX1 overexpression. (B) The relative expression levels of CDK2 and PCNA protein after CUX1 overexpression. (C) OD450 values in the CCK8 assay after CUX1 overexpression. (D) Cell-cycle analysis of Hu sheep DPCs overexpressing CUX1 by flow cytometry. (E) The number of proliferating Hu sheep DPCs after CUX1 overexpression was detected by the EdU assay. EdU staining (red) indicates proliferating cells; Hoechst staining (blue) indicates nuclei. (F) The proportion of EdU-positive Hu sheep DPCs. (G) The rate of different periods of the cell cycle * p < 0.05, ** p < 0.01.
Figure 5
Figure 5
CUX1 knockdown inhibits proliferation of Hu sheep DPCs. (A) mRNA expression levels of PCNA, CYCLIN D1, and CDK2 after CUX1 knockdown. (B) The relative expression levels of CDK2 and PCNA protein after CUX1 knockdown. (C) OD450 values in the CCK8 assay after CUX1 knockdown. (D,G) Cell-cycle analysis of CUX1-silenced Hu sheep DPCs by flow cytometry. (E) The number of proliferating Hu sheep DPCs after CUX1 knockdown was detected by the EdU assay. EdU staining (red) indicates proliferating cells; Hoechst staining (blue) indicates nuclei. (F) The proportion of EdU-positive Hu sheep DPCs. (G) The rate of different periods of the cell cycle * p < 0.05, ** p < 0.01.
Figure 6
Figure 6
Expression of Wnt/β-catenin signaling pathway genes after CUX1 overexpression or knockdown. (A) mRNA expression levels of different genes after CUX1 overexpression. (B) mRNA expression levels of different genes after CUX1 knockdown. * p < 0.05, ** p < 0.01.
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