KDM6B Negatively Regulates the Neurogenesis Potential of Apical Papilla Stem Cells via HES1

Abstract

Stem cells from the apical papilla (SCAPs) are used to regulate the microenvironment of nerve defects. KDM6B, which functions as an H3K27me3 demethylase, is known to play a crucial role in neurogenesis. However, the mechanism by which KDM6B influences the neurogenesis potential of SCAPs remains unclear. We evaluated the expression of neural markers in SCAPs by using real-time RT-PCR and immunofluorescence staining. To assess the effectiveness of SCAP transplantation in the SCI model, we used the BBB scale to evaluate motor function. Additionally, toluidine blue staining and Immunofluorescence staining of NCAM, NEFM, β-III-tubulin, and Nestin were used to assess nerve tissue remodeling. Further analysis was conducted through Microarray analysis and ChIP assay to study the molecular mechanisms. Our results show that KDM6B inhibits the expression of NeuroD, TH, β-III tubulin, and Nestin. In vivo studies indicate that the SCAP-KDM6Bsh group is highly effective in restoring spinal cord structure and motor function in rats suffering from SCI. Our findings suggest that KDM6B directly binds to the HES1 promoter via regulating H3K27me3 and HES1 expression. In conclusion, our study can help understand the regulatory role of KDM6B in neurogenesis and provide more effective treatments for nerve injury.

Keywords: hairy and enhancer of split 1 (HES1); lysine (K)-specific demethylase 6B (KDM6B); neurogenesis potential; stem cells from apical papilla (SCAPs).

Figure 1

KDM6B knockdown enhanced the expression of neural markers in SCAPs. (A) The efficiency of KDM6B knockdown was tested by Western blot, with Histone H3 serving as an internal control; (B,C) compared with the control group, KDM6B knockdown increased the volume of neurospheres, demonstrated by a scale bar of 100 μm; (D–F) real-time RT-PCR analysis revealed that KDM6B knockdown significantly upregulated the expression of NeuroD (D), TH (E), and β-III tubulin (F) in SCAPs, with GAPDH as an internal control; and (G–J) immunofluorescence staining indicated that the expression levels of Nestin and β-III Tubulin, as well as the percentage of Nestin and β-III Tubulin positive cells in neurospheres, were also elevated in the KDM6Bsh group compared with the control group. Statistical significance was determined using Student’s t-test, and all error bars represent SD (n = 3). * p < 0.05. ** p ≤ 0.01.

Figure 2

KDM6B overexpression inhibited the expression of neural markers in SCAPs. (A) The KDM6B overexpression efficiency was tested by Western blot, with Histone H3 serving as an internal control; (B,C) compared with the control group, KDM6B overexpression decreased the volume of neurospheres, demonstrated by a scale bar of 100 μm; (D–F) real-time RT-PCR analysis revealed that KDM6B overexpression significantly downregulated the expression of NeuroD (D), TH (E), and β-III tubulin (F) in SCAPs, with GAPDH as an internal control; and (G–J) immunofluorescence staining indicated that the expression levels of Nestin and β-III tubulin, as well as the percentage of Nestin and β-III tubulin positive cells in neurospheres, were also decreased in the HA-KDM6B group compared with the control group. Statistical significance was determined using Student’s t-test, and all error bars represent SD (n = 3). * p < 0.05. ** p ≤ 0.01.

Figure 3

The morphological and functional changes after SCAP transplantation in SCI model. (A) The limb motor function of rats was assessed using the BBB scale at 1–5 weeks post-SCI; (B) compared with the Scramsh group, the KDM6Bsh group exhibited a gradual improvement in spinal function recovery from week 3 to 5, as demonstrated by the BBB scale; and (C) the spinal cord morphology and histological structures of the KDM6Bsh group were found to be better recovered compared with those of the SCI and Scramsh groups. The yellow arrows depict regions of spinal cord injury. Statistical significance was determined using the one-way ANOVA and Kruskal–Wallis test and all error bars represent SD (n = 6). * p < 0.05.

Figure 4

The pathological changes after SCAP transplantation in SCI model. (A) HE staining result revealed that the KDM6Bsh group had fewer cavities and scars compared with the SCI and Scramsh group. Scale bar, 100 μm, and 500 μm; (B) toluidine blue staining result revealed that obvious tissue cavities were observed in the SCI group, and the KDM6Bsh group showed very fine cavities compared with the Scramsh group; and (C) the immunofluorescence results indicated that h-mitochondria-positive cells were in the injury area of Scramsh and KDM6Bsh groups. Scale bar, 50 μm.

Figure 5

The neural marker expression after SCAP transplantation in SCI model. (A) The immunofluorescence staining of NCAM and NEFM. Scale bar, 500 μm; (B) statistical analysis of the percentage of NCAM in the lesion zone in four groups; (C) statistical analysis of the percentage of NEFM in the lesion zone in four groups; (D) the immunofluorescence staining of Nestin and β-III tubulin. Scale bar, 500 μm; (E) statistical analysis of the percentage of Nestin in the lesion zone in four groups; and (F) statistical analysis of the percentage of β-III tubulin in the lesion zone in four groups. Statistical significance was determined using the one-way ANOVA and Kruskal–Wallis test. ns: no significant difference. ** p ≤ 0.01.

Figure 6

Significant gene ontology (GO) analyses of differentially expressed genes in KDM6B overexpressed SCAPs compared with the control group. The GO functions were represented by the x-axis, while the y-axis indicated the percentage and number of genes associated with each function. The results revealed that there were distinct GO functions associated with upregulated (A) and downregulated genes (B).

Figure 7

Statistics of KEGG pathway enrichment of differentially expressed genes in KDM6B overexpressed SCAPs compared with the control group. The statistics of KEGG pathway enrichment of upregulated genes (A) and downregulated genes (B) during KDM6B overexpression. The statistical analysis is presented in circular plots, where each circle represents a KEGG pathway. The number of annotated genes for different KEGG terms is indicated by circular dots, while different colors represent various levels of significance (p-value). The rich factor, which reflected the ratio of gene numbers for each term to the numbers of all genes with terms, is also provided.

Figure 8

Knockdown of KDM6B directly increases the H3K27me3 enrichment in the HES1 promoter. (A–C) Real-time RT-PCR analysis was conducted on the HA-KDM6B group, which showed a significant reduction in the expression of HES1 (A), EGR1 (B), and NR4A2 (C). (D–F) Real-time RT-PCR analysis was performed on SCAPs, revealing an upregulation of HES1 (D), EGR1 (E), and NR4A2 (F) expression. GAPDH was used as an internal control. (G,H) ChIP experiment results revealed that KDM6B depletion increased the enrichment of H3K27me3 in the HES1 promoter. Statistical significance was determined using Student’s t-test, with all error bars representing SD (n = 3). * p < 0.05 and ** p ≤ 0.01.