Abstract：
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.
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.
摘要：
RUNX2（Runt相关转录因子2）在人牙髓干细胞（hDPSC）的牙源性分化中起关键调节因子的作用。此外，外显子5的包含对于RUNX2功能是重要的。我们先前的研究表明Y-盒结合蛋白1(YBX1)促进RUNX2外显子5包合和hDPSC的矿化。然而,RUNX2外显子5选择性剪接的调控机制有待进一步探索。
通过RT-PCR和Westernblot分析了hDPSCs牙源性分化过程中异质核核糖核蛋白A1（hnRNPA1）的表达水平。通过功能增益和功能丧失实验分析了hnRNPA1在RUNX2外显子5的可变剪接和牙间充质细胞的牙源性分化中的作用。
令人惊讶的是,我们发现了一个可变的剪接因子,hnRNPA1在调节hDPSC的RUNX2外显子5包合和牙源性分化中具有与YBX1相反的作用。通过功能增益和丧失试验，我们发现hnRNPA1抑制了RUNX2外显子5的包含，从而抑制了成牙本质细胞的分化。hnRNPA1过表达可抑制碱性磷酸酶(ALP)和骨钙蛋白(OCN)的表达,以及在hDPSC和小鼠牙乳头细胞（mDPC）的牙源性分化过程中矿化结节的形成，而使用hnRNPA1敲除制剂获得了相反的结果。
本研究表明hnRNPA1抑制RUNX2外显子5包合，降低hDPSC和mDPC的牙源性分化能力。
通过RT-PCR和Westernblot分析了hDPSCs牙源性分化过程中异质核核糖核蛋白A1（hnRNPA1）的表达水平。通过功能增益和功能丧失实验分析了hnRNPA1在RUNX2外显子5的可变剪接和牙间充质细胞的牙源性分化中的作用。
令人惊讶的是,我们发现了一个可变的剪接因子,hnRNPA1在调节hDPSC的RUNX2外显子5包合和牙源性分化中具有与YBX1相反的作用。通过功能增益和丧失试验，我们发现hnRNPA1抑制了RUNX2外显子5的包含，从而抑制了成牙本质细胞的分化。hnRNPA1过表达可抑制碱性磷酸酶(ALP)和骨钙蛋白(OCN)的表达,以及在hDPSC和小鼠牙乳头细胞（mDPC）的牙源性分化过程中矿化结节的形成，而使用hnRNPA1敲除制剂获得了相反的结果。
本研究表明hnRNPA1抑制RUNX2外显子5包合，降低hDPSC和mDPC的牙源性分化能力。
