长链非编码RNA CALB2上调GADD34表达促进牙髓干细胞增殖和成牙本质向分化的机制研究

Dental pulp stem cells (DPSCs) play an important role in the repair and regeneration of dental pulp. In our preliminary studies, we applicated the lncRNAs microarray technology to detect and screen the specific lncRNAs in odontoblast differentiation of DPSCs. We found that lncRNA CALB2 was significantly increased in odontoblast differentiation of DPSCs. Over-expression of lncRNA CALB2 can significantly promote the proliferation and odontoblast differentiation of DPSCs. We used RNA pull-down experiments to screen and found that the lncRNA CALB2 bound with the protein GADD34 that can activate Wnt signal pathway. We applicated bioinformatics analysis and showed that miR-221 target the binding sites of lncRNA CALB2 in the 3’UTR of GADD34. And we found that the endogenous GADD34 of the cell interacted with PP1 and Axin. Therefore, we propose that lncRNA CALB2 can mediate de-repression of GADD34 by blocking miR-221, leading to GADD34 upregulation, and mediating PP1 dephosphorylation of Axin, which abnormally activating of Wnt/β-catenin signaling pathway. We intend to further confirm this event and discuss 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成牙本质向分化过程中特异表达的LncRNA CALB2，上调表达LncRNA CALB2能促进DPSCs增殖和成牙本质向分化； RNA pull-down实验筛选到LncRNA CALB2与激活Wnt通路的蛋白质GADD34相结合；生物信息学发现GADD34 3′UTR中miR-221的结合位点与LncRNA CALB2的结合序列有重叠；并且GADD34介导PP1与Axin的相互作用。因此，我们提出LncRNA CALB2能竞争miR-221结合GADD34，解除靶向抑制，上调表达的GADD34介导PP1使Axin去磷酸化，激活Wnt/β-catenin信号通路。本项目拟进一步证实这一事件并探讨其对DPSCs成牙本质向分化的重要性和意义，为临床修复牙髓损伤提供新的思路。

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。DPSCs 增殖和成牙本质向分化是牙髓损伤修复和治疗的关键环节。RUNX2在骨形成过程中起关键作用，并且是成骨过程中最主要的调节剂之一。成牙本质向分化与成骨分化有相似的发育过程。本项目采用LncRNA芯片筛选出组织矿化液诱导DPSCs成牙本质向分化相关的LncRNA CALB2。根据生物信息学DIANA工具和HMDD分析的结果，筛选了3个直接靶向LncRNA CALB2和RUNX2的 miRNA，分别为miR-30b-3p，miR-148-5p和miR-192-3p。上调表达LncRNA CALB2能促进DPSCs成牙本质向分化。通过生物信息学和双荧光素酶报告基因检测，充分证实了LncRNA CALB2为miR-30b-3p的分子海绵，且RIP实验结果证明LncRNA CALB2存在于含Ago2的miRNP中，可与miR-30b-3p相互作用。同时验证了LncRNA CALB2可以通过竞争miR-30b-3p来调节DPSCs细胞成牙本质向分化。并充分证实了miR-30b-3p直接靶向调节RUNX2。进而阐明了LncRNA CALB2 / miR-30b-3p / RUNX2轴调节DPSCs细胞成牙本质向分化。本项目为研究牙髓牙本质复合体的再生提供了重大的契机，将为“组织工程学修复牙髓损伤”这一课题向临床转化提供新的思路。