LncRNA DANCR调控miR-216a/c-Cbl轴在牙髓防御修复中的作用研究

Enhanced odontoblast differentiation of human dental pulp cells (DPCs) is considered a keystone in dentin-pulp complex formation. In our published paper and preliminary studies, we identified lncRNA DANCR was downregulated during the differentiation process of DPCs and the reparative response of dental pulp, while overexpression of DANCR suppressed odontoblast differentiation of DPCs by inhibiting Wnt/β-catenin signaling. Notably, we further provided evidence that DANCR could competitively bind to miR-216a and there was a negative correlation between DANCR and miR-216a expression during the differentiation process. Moreover, recent studies reported that c-Cbl, the target gene of miR-216a, inactivated Wnt signaling pathway. Therefore, we hypothesized that DANCR may act as a competing endogenous RNA to bind miR-216a, then stabilize c-Cbl protein translation, thereby inhibiting Wnt signaling pathway and odontoblast differentiation. In this grant, we will utilize RIP and luciferase reporter to explore the relationship among DANCR/miR-216a/c-Cbl, and clarify the significance of this event in regulation of Wnt signaling and the odontoblast differentiation of DPCs. Moreover, the effect of DANCR downexpression in DPCs differentiation and dental pulp-dentin complex formation will be accessed through in vivo experiment, which will be conducted by transplanting root segments subcutaneously into immune deficiency mice. Our results will provide new ideas for further study and clinical translation of the reparative response of dental pulp.

促进牙髓细胞（DPCs）成牙本质向分化是牙髓牙本质复合体再生的关键。本人已发表论文与预实验结果发现：在DPCs成牙本质向分化及牙髓组织修复性反应中，lncRNA DANCR表达下调，如高表达，则可阻遏Wnt/β-catenin通路抑制DPCs分化；DANCR与miR-216a结合，且在分化过程中呈负相关，而miR-216a靶向沉默c-Cbl，后者抑制Wnt通路。因此我们提出：DANCR可竞争性结合miR-216a，解除对c-Cbl的靶向沉默，进而抑制Wnt通路调控DPCs成牙本质向分化。本项目拟进一步运用RIP、报告基因等技术分析DANCR/miR-216a/c-Cbl的相互关系，探讨这种竞争调控模式对下游Wnt通路及DPCs成牙本质向分化的生物学效应；采用裸鼠皮下移植实验，阐明敲低DANCR对DPCs成牙本质向分化及牙髓牙本质复合体形成的促进作用，为牙髓防御修复研究及转化应用提供新思路。

当牙齿受到外来刺激时，牙髓组织会启动自我防御反应，促进hDPCs成牙本质向分化，形成修复牙本质保护牙髓。因此，探索如何调控这群异质性细胞群，充分发挥其成牙本质向分化潜能以达到防御和修复牙本质的目的，是口腔再生医学领域的研究重点。长链非编码 RNA（lncRNA）可从转录水平、转录后水平、表观遗传学等多个层面调控生物学过程。近年来，lncRNAs在胚胎或成体细胞定向分化和自我更新中的作用受到广泛关注。本研究前期研究提示lncRNA DANCR可作为修复牙髓损伤的潜在治疗靶点。然而，lncRNA DANCR在hDPCs成牙本质向分化中的调控机制尚不清楚。本课题开展系列研究，验证了长链非编码RNA通过调控牙髓细胞分化进而参与牙髓组织损伤修复再生，同时也进一步拓展研究了牙髓干细胞在牙髓组织损伤修复中的作用。获得结果如下：①为了探讨lncRNA DANCR在hDPCs成牙本质向分化中的作用。通过低表达lncRNA DANCR发现其促进hDPCs分化。生物信息分析证实lncRNA DANCR与miR-216a结合，且两者表达负相关。lncRNA DANCR通过直接结合抑制miR-216a发挥其生物学作用，而miR-216a可靶向沉默c-Cbl蛋白。与此同时，敲低miR-216a可以解除对c-Cbl的抑制作用，并逆转低表达lncRNA DANCR对hDPCs分化的影响。②为了探究牙髓干细胞在牙髓组织损伤修复中的作用。我们发现牙髓干细胞在不同牙髓组织微环境下呈现多方面特性，可作为：1)修复细胞，2)炎性细胞，3)免疫调节细胞。这种免疫调节特性由局部微环境决定，因此，基于细胞的治疗策略应用于炎症组织的修复和再生时，应考虑到干细胞的多方面作用。相关结果探讨了牙髓细胞作为组织损伤修复和再生种子细胞的可行性，为组织工程牙髓再生和活髓保存治疗提供新的视角。