LncRNA DANCR sponges miR-216a to inhibit odontoblast differentiation through upregulating c-Cbl
LncRNA DANCR sponges miR-216a to inhibit odontoblast differentiation through upregulating c-Cbl
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LncRNA DANCR 海绵 miR-216a 通过上调 c-Cbl 抑制成牙本质细胞分化
DOI：
10.1016/j.yexcr.2019.111751
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发表时间：
2020-02-01
影响因子：
3.7
通讯作者：
Lin, Zhengmei
中科院分区：
文献类型：
--
作者：
Chen, Lingling;Song, Zhi;Lin, Zhengmei
人牙髓细胞（human dental pulp cells，hDPC）成牙本质细胞分化的增强被认为是牙本质-牙髓复合体形成的关键。我们已经发现lncRNA DANCR参与了hDPC的分化过程，然而，其在hDPC成牙本质细胞分化中的作用机制仍有待进一步研究。在本研究中，我们采用功能丧失的方法，确定了DANCR的下调驱动hDPC的成牙本质细胞分化。生物信息学分析表明，DANCR中含有miR-216a的结合位点，且DANCR与miR-216a呈负相关。采用双荧光素酶报告基因检测和RNA结合蛋白免疫沉淀（RIP）技术进一步证实DANCR通过与miR-216 a直接结合发挥功能。值得注意的是，miR-216 a能够结合c-Cbl的3'-UTR并抑制其表达。此外，c-Cbl蛋白表达水平在hDPC分化过程中显著下调，而c-Cbl过表达抑制hDPC向成牙本质细胞分化。下调miR-216 a可有效逆转DANCR基因敲低对c-Cbl水平和成牙本质细胞分化的抑制作用。综上所述，这些分析表明，DANCR通过海绵状miR-216 a正向调节c-Cbl的表达，并抑制hDPC的成牙本质细胞分化。我们的研究结果将扩大再生医学中基于细胞的治疗的临床应用领域。
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.