The State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Key Laboratory of Oral Biomedicine Ministry of Education, Hubei Key Laboratory of Stomatology, School & Hospital of Stomatology, Wuhan University, 430079 Wuhan, Hubei, China.
Department of Endodontics, School & Hospital of Stomatology, Wuhan University, 430079 Wuhan, Hubei, China.
Front Biosci (Landmark Ed). 2023 Jul 19;28(7):139. doi: 10.31083/j.fbl2807139.
RUNX2 (Runt-related transcription factor 2) acts as a key regulator in the odontogenic differentiation of human dental pulp stem cells (hDPSCs). Moreover, the inclusion of exon 5 is important for RUNX2 function. Our previous study showed that Y-Box Binding Protein 1 (YBX1) promoted RUNX2 exon 5 inclusion and mineralization of hDPSCs. However, the regulatory mechanism of RUNX2 exon 5 alternative splicing needed further exploration.
The expression level of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) during the odontogenic differentiation of hDPSCs was analyzed by RT-PCR and Western blot. The roles of hnRNP A1 in the alternative splicing of RUNX2 exon 5 and the odontogenic differentiation of dental mesenchymal cells were analyzed by gain- and loss-of-function experiments.
Surprisingly, we found an alternative splicing factor, hnRNP A1, which had an opposite role to YBX1 in regulating RUNX2 exon 5 inclusion and odontogenic differentiation of hDPSCs. Through gain- and loss-of-function assay, we found that hnRNP A1 suppressed the inclusion of RUNX2 exon 5, resulting in the inhibition of odontoblastic differentiation. The overexpression of hnRNP A1 can inhibit the expression of ALP (alkaline phosphatase) and OCN (osteocalcin), and the formation of mineralized nodules during the odontogenic differentiation of both hDPSCs and mouse dental papilla cells (mDPCs), whereas the opposite results were obtained with an hnRNP A1 knockdown preparation.
The present study indicated that hnRNP A1 suppressed RUNX2 exon 5 inclusion and reduced the odontogenic differentiation ability of hDPSCs and mDPCs.
RUNX2（Runt 相关转录因子 2）作为一种关键调节因子，在人牙髓干细胞（hDPSCs）的牙源性分化中起作用。此外，外显子 5 的包含对于 RUNX2 功能很重要。我们之前的研究表明，Y 盒结合蛋白 1（YBX1）促进 hDPSCs 的 RUNX2 外显子 5 包含和矿化。然而，RUNX2 外显子 5 选择性剪接的调节机制需要进一步探索。
通过 RT-PCR 和 Western blot 分析 hDPSCs 牙源性分化过程中异质核核糖核蛋白 A1（hnRNP A1）的表达水平。通过获得和丧失功能实验分析 hnRNP A1 在 RUNX2 外显子 5 选择性剪接和牙间质细胞牙源性分化中的作用。
令人惊讶的是，我们发现一种剪接因子 hnRNP A1，它在调节 RUNX2 外显子 5 包含和 hDPSCs 牙源性分化方面与 YBX1 作用相反。通过获得和丧失功能实验，我们发现 hnRNP A1 抑制 RUNX2 外显子 5 的包含，从而抑制成牙本质细胞分化。hnRNP A1 的过表达可以抑制碱性磷酸酶（ALP）和骨钙素（OCN）的表达，以及 hDPSCs 和小鼠牙乳头细胞（mDPCs）牙源性分化过程中矿化结节的形成，而 hnRNP A1 敲低制剂则得到相反的结果。
本研究表明 hnRNP A1 抑制 RUNX2 外显子 5 的包含，降低 hDPSCs 和 mDPCs 的牙源性分化能力。
