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NAT10 promotes osteogenic differentiation of periodontal ligament stem cells by regulating VEGFA-mediated PI3K/AKT signaling pathway through ac4C modification

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Abstract

Periodontal tissue regeneration engineering based on human periodontal ligament stem cells (hPDLSCs) provides a broad prospect for the treatment of periodontal disease. N-Acetyltransferase 10 (NAT10)-catalyzed non-histone acetylation is widely involved in physiological or pathophysiological processes. However, its function in hPDLSCs is still missing. hPDLSCs were isolated, purified, and cultured from extracted teeth. Surface markers were detected by flow cytometry. Osteogenic, adipogenic, and chondrogenic differentiation potential was detected by alizarin red staining (ARS), oil red O staining, and Alcian blue staining. Alkaline phosphatase (ALP) activity was assessed by ALP assay. Quantitative real-time PCR (qRT-PCR) and western blot were used to detect the expression of key molecules, such as NAT10, Vascular endothelial growth factor A (VEGFA), PI3K/AKT pathway, as well as bone markers (RUNX2, OCN, OPN). RNA-Binding Protein Immunoprecipitation PCR (RIP-PCR) was used to detect the N4-acetylcytidine (ac4C) mRNA level. Genes related to VEGFA were identified by bioinformatics analysis. NAT10 was highly expressed in the osteogenic differentiation process with enhanced ALP activity and osteogenic capability, and elevated expression of osteogenesis-related markers. The ac4C level and expression of VEGFA were obviously regulated by NAT10 and overexpression of VEGFA also had similar effects to NAT10. The phosphorylation level of PI3K and AKT was also elevated by overexpression of VEGFA. VEGFA could reverse the effects of NAT10 in hPDLSCs. NAT10 enhances the osteogenic development of hPDLSCs via regulation of the VEGFA-mediated PI3K/AKT signaling pathway by ac4C alteration.

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Data availability

All data generated or analysed during this study are included in this published article.

Abbreviations

hPDLSCs:

Human periodontal ligament stem cells

NAT10:

N-Acetyltransferase 10

ALP:

Alkaline phosphatase

qRT-PCR:

Quantitative real-time PCR

VEGFA:

Vascular endothelial growth factor A

RIP-PCR:

RNA-Binding Protein Immunoprecipitation PCR

ac4C:

N4-Acetylcytidine

MSCs:

Mesenchymal stem cells

ARS:

Alizarin red staining

SD:

Standard deviation

ANOVA:

One-way analysis of variance

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Acknowledgements

We would like to give our sincere gratitude to the reviewers for their constructive comments.

Funding

This work was supported by the role of ETS2-NAT10 in promoting osteogenic differentiation of BMSCs by activating PRP (2022JC085).

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Authors and Affiliations

  1. Pediatric Surgery, Children’s Hospital of Changchun, Changchun, 130021, Jilin Province, People’s Republic of China

    Zhao Cui, Peng Wu, Ying Lu, Yongxin Tao, Chuibing Zhou, Ruting Cui & Jingying Li

  2. Department of Stomatology, The First Hospital of Jilin University, Changchun, 130021, Jilin Province, People’s Republic of China

    Yunhe Xu

  3. Pediatric Surgery, Children’s Hospital of Changchun, No. 1321, Beian Road, Chaoyang District, Changchun, 130021, Jilin Province, People’s Republic of China

    Rongpeng Han

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  1. Zhao Cui
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10266_2023_793_MOESM1_ESM.tif

Supplementary file 1: Fig. S1. Mutated NAT10 reduced ac4C level and inhibits osteogenesis. A. RIP was conducted to detect the ac4C level in the control and NAT10-KR group. B. Western blot was conducted to detect Runx2, OCN, and OPN. There were more than three of these duplicates. Mean ± SD was used to express the data. *P <0.05, **P< 0.01, and ***P< 0.001 are all considered significant.

10266_2023_793_MOESM2_ESM.tif

Supplementary file 2: Fig. S2. NAT10 stimulates the phosphorylation of PI3K and AKT. A. Western blot was performed to detect the expression of p-PI3K, PI3K, p-AKT, and AKT. There were more than three of these duplicates. Mean ± SD was used to express the data. *P <0.05, **P< 0.01, and ***P< 0.001 are all considered significant.

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Cui, Z., Xu, Y., Wu, P. et al. NAT10 promotes osteogenic differentiation of periodontal ligament stem cells by regulating VEGFA-mediated PI3K/AKT signaling pathway through ac4C modification. Odontology 111, 870–882 (2023). https://doi.org/10.1007/s10266-023-00793-1

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