LncRNA PVT1上调Lin28表达在牙髓干细胞增殖和成牙本质向分化中的作用及机制研究

Dental pulp stem cells (DPSCs) play an important role in the repair and regeneration of dental pulp. In this study, we investigated lncRNAs alterations that contribute to odontoblast differentiation of DPSCs through microarray expression profiling. Here, we report that lncRNA PVT1 is a key regulator of the odontoblast differentiation of DPSCs. Over-expression of lncRNA PVT1 can significantly promote the proliferation and odontoblast differentiation of DPSCs. Furthermore, using RNA pull-down and mass spectrometry, we found that the lncRNA PVT1 bound with the Lin28 protein, a key molecule of regulating proliferation and differentiation of stem cell. We applied bioinformatics methods and found the same sequence alignment binding with miR-128 and miR-203 in lncRNA PVT1 and Lin28 3'-UTR sequence. Therefore, we propose that lncRNA PVT1 acts as a ceRNA to sponge for miR-128 and miR-203 to stabilize Lin28 mRNA and promote protein translation, leading to increase the Lin28 expression. We intend to further confirm this event and explore its importance and significance for proliferation and odontoblast differentiation of DPSCs, which would provide new ideas for clinical repair of pulp injury.

牙髓干细胞（DPSCs）在牙髓损伤修复和再生过程中发挥重要作用。我们前期应用LncRNAs芯片筛选到DPSCs成牙本质向分化相关的LncRNA PVT1，其上调表达能促进DPSCs增殖和成牙本质向分化；应用RNA pull-down实验及质谱鉴定出LncRNA PVT1与调控干细胞增殖和定向分化的关键蛋白分子Lin28结合；生物信息学分析发现LncRNA PVT1与Lin28 3'UTR序列存在共同结合miR-128, miR-203的位点。因此，我们提出LncRNA PVT1作为CeRNA，竞争miR-128, miR-203结合Lin28 3'UTR，解除二者对Lin28的靶向调控，稳定Lin28 mRNA和促进蛋白翻译，上调Lin28蛋白的表达。本项目拟进一步阐明这一事件并探讨其对DPSCs增殖和成牙本质向分化的重要性及意义，为临床修复牙髓损伤提供新的思路。

龋齿引起牙髓损伤，人牙髓干细胞（DPSCs）会增殖并迁移到损伤部位，然后分化为成牙母细胞，形成修复性牙本质。因此，DPSCs被认为是再生牙髓病学的有前途的细胞。然而，DPSCs成牙本质向分化的机制仍不清楚。在这里，（1）我们应用LncRNAs芯片筛选到DPSCs成牙本质向分化相关的LncRNA PVT1，高表达LncRNA PVT1能促进DPSCs增殖和成牙本质向分化。（2）我们应用RNA pull-down实验及质谱鉴定出LncRNA PVT1与调控干细胞增殖和定向分化的关键蛋白分子Lin28结合，且RIP实验显示LncRNA PVT1能被Lin28抗体富集，充分证实LncRNA PVT1与蛋白质Lin28结合。（3）生物信息学分析表明LncRNA PVT1与Lin28 3'UTR序列存在共同结合miR-128, miR-203的位点，采用双荧光素酶报告基因测定证实，lncRNA PVT1可以直接与miR-128, miR-203结合，即LncRNA PVT1竞争miR-128, miR-203结合Lin28 3'UTR，进行转录后调控Lin28蛋白的表达。（4）LncRNA PVT1高表达显著上调Lin28、DSPP和DMP-1的蛋白水平，而敲低Lin28表达可削除了这一作用。因此，这些发现表明，一方面，LncRNA PVT1可直接结合Lin28蛋白并稳定其蛋白表达，另一方面，LncRNA PVT1作为CeRNA，竞争miR-128, miR-203结合Lin28 3'UTR，解除二者对Lin28的靶向调控，稳定Lin28 mRNA进而上调Lin28蛋白的表达。综上，LncRNA PVT1 / miR-128, miR-203 / Lin28信号轴可能是促进DPSCs增殖和成牙本质向分化的新治疗靶点，为临床修复牙髓损伤提供了新的思路。