Enhanced odontoblast differentiation of human dental pulp cells (hDPCs) is considered a keystone in dentin-pulp complex formation. We have revealed lncRNA DANCR was implicated in this differentiation program, however, its mechanism in odontoblast differentiation of hDPCs remains further explored. In this study, by employing loss-of-function approach, we identified downregulation of DANCR drived odontoblast differentiaion of hDPCs. Bioinformatics analysis was utilized to show that DANCR contained binding site for miR-216a and an inverse correlation between DANCR and miR-216a was obtained. Dual luciferase reporter assay and RNA-binding protein immunoprecipitation (RIP) were applied to further confirm that DANCR conferred its functions by directly binding to miR-216a. Notably, miR-216a was able to bind to the 3'-UTR of c-Cbl and repressed its expression. In addition, the protein level of c-CBL was significantly downregulated during hDPCs differentiation, while c-Cbl overexpression inhibited odontoblast differentiation of hDPCs. Moreover, downregulation of miR-216a efficiently reversed the suppression of c-Cbl level and odontoblast differentiation induced by knockdown of DANCR. Taken together, these analyses indicated that DANCR positively regulated the expression of c-Cbl, through sponging miR-216a, and inhibited odontoblast differentiation of hDPCs. Our results will extend the field of clinical application for cell-based therapy in regenerative medicine.

中文翻译：

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LncRNA DANCR使miR-216a海绵通过上调c-Cbl抑制成牙本质细胞分化。

人牙髓细胞（hDPC）的成牙本质细胞分化增强被认为是牙本质-牙髓复合物形成的基石。我们已经揭示了lncRNA DANCR与该分化程序有关，但是，其在hDPC的成牙本质细胞分化中的机制仍有待进一步探索。在这项研究中，通过采用功能丧失方法，我们确定了DANCR驱动的hDPC的成牙本质细胞分化的下调。利用生物信息学分析表明，DANCR含有miR-216a的结合位点，并且获得了DANCR和miR-216a的反相关关系。应用双重荧光素酶报告基因测定法和RNA结合蛋白免疫沉淀法（RIP）进一步证实DANCR通过直接与miR-216a结合而赋予了其功能。值得注意的是，miR-216a能够与3' c-Cbl的-UTR并抑制其表达。另外，在hDPCs分化过程中，c-CBL的蛋白水平显着下调，而c-Cbl的过表达抑制了hDPCs的成牙本质细胞分化。而且，miR-216a的下调有效地逆转了由DANCR敲低诱导的c-Cbl水平抑制和成牙本质细胞分化。综上，这些分析表明，DANCR通过使miR-216a海绵化，正向调节c-Cbl的表达，并抑制hDPC的成牙本质细胞分化。我们的结果将扩展再生医学中基于细胞的治疗的临床应用领域。miR-216a的下调有效地逆转了DANCR敲低诱导的c-Cbl水平抑制和成牙本质细胞分化。综上，这些分析表明，DANCR通过使miR-216a海绵化，正调控c-Cbl的表达，并抑制hDPC的成牙本质细胞分化。我们的结果将扩展再生医学中基于细胞的治疗的临床应用领域。miR-216a的下调有效地逆转了DANCR敲低诱导的c-Cbl水平抑制和成牙本质细胞分化。综上，这些分析表明，DANCR通过使miR-216a海绵化，正调控c-Cbl的表达，并抑制hDPC的成牙本质细胞分化。我们的结果将扩展再生医学中基于细胞的治疗的临床应用领域。