Klf4相关的CeRNA与成牙本质细胞分化

CeRNA（competing endogenous RNA）is a kind of endogenous RNAs that share multiple MREs（MicroRNA response elements）and regulate their targets in a MicroRNA competing based mechanism.In this project, Klf4 related CeRNAs were predicted by a serial of bioinformatical methods. And further preliminary study also confirmed existence of these CeRNAs. To explore the function of KLF4 related CeRNA network in odontoblastic differentiation, following measures will be applied. Firstly, in situ hybridization and qPCR technology will be used to detect the expression pattern of Klf4 related CeRNAs during odontoblastic differentiation both in vivo and in vitro. Then, CeRNA of Klf4 will be overexpressed as well as knockdown in odontoblast like cell line, and biologic effects of 3'UTR of CeRNA on Klf4 will be investigated by the evaluation of mRNA level, protein level and luciferase activities. To confirm the microRNA dependency of KLF4 related CeRNA network, a Dicer knockout mouse dental papilla cell line will be established. And biologic effects of CeRNA on Klf4 is consider to be compromised in this cell line. Also, the effect of the shared microRNA on Klf4, CeRNA and odontoblastic differentiation will be investigated. After certification, function of Klf4 related CeRNA on odontoblastic differentiation will be determined by CeRNA overexpression and knockdown in vitro. In the end, CeRNA 3'UTR conditional overexpression transgenic mice will be established to confirm the function of CeRNA in vivo. This project is a succession of the past two NSCF (NO.81070797 and 81070797).And the achievements of this project will enforce the interpretation of the regulation of odontoblastic differentiation.

CeRNA是一种通过竞争MicroRNA而调控靶基因的竞争性内源RNA。为了研究Klf4相关的CeRNA在成牙本质细胞分化过程中的作用,项目组前期采用生物信息学预测出Klf4相关CeRNA，并使用RNA免疫共沉淀、过表达等技术对这些CeRNA进行了初步的验证。后期拟采用原位杂交和qPCR等方法确认Klf4与其CeRNA在成牙本质细胞分化中的共表达。继而，过表达或抑制CeRNA ，检测CeRNA对Klf4的调控作用。构建Dicer敲除的小鼠牙乳头细胞系，在其中验证CeRNA对Klf4调控的MicroRNA依赖性。运用过表达和miRNA干扰探针研究共享MicroRNA对Klf4与其CeRNA的调控作用。最后，通过体外过表达或抑制CeRNA，以及体内构建CeRNA的3'UTR条件性过表达小鼠，研究CeRNA对成牙本质细胞分化的调控作用。本项目研究成果将加深对调控成牙本质细胞分化的分子机制的理解。

成牙本质细胞分化是一种非常独特且复杂的过程，有很多种分子机制参与调控，包括转录因子，信号分子和以microRNA为代表的表观调控因子等。这些分子相互联系形成网络并从不同层面上调节成牙本质细胞分化。本项目主要研究Klf4相关的ceRNA网络在成牙本质细胞分化过程中的功能。项目组采用原位杂交和qRT-PCR等方法检测前期初步验证的Klf4的CeRNA在成牙本质细胞体内及体外分化中的表达特征，如Sp1，Qki及Tnrc6b等，确认Klf4与其CeRNA的共表达。继而，过表达或抑制ceRNA的3’UTR，检测ceRNA对Klf4的调控作用，反正亦然。运用siRNA技术敲低Dicer或利用双荧光素酶实验，验证ceRNA对Klf4调控具有microRNA依赖性。运用microRNA mimics转染成牙本质细胞，通过western blotting技术检测共享micro RNA对Klf4与其ceRNA的调控作用，并利用双荧光素酶实验证明其确实能够结合。本项目研究结果证明Sp1、Qki等基因的mRNA作为Klf4 的ceRNA 能够通过与Klf4竞争性的结合microRNA 来上调Klf4的表达，从而促进成牙本质细胞分化。最后项目组检测了ceRNA对成牙本质细胞分化的调控作用。分别上调或者抑制ceRNA的表达，观察其对成牙本质向分化过程的影响。此外，项目组还观察到Sp1，Qki还可分别通过其他方式，如结合到Dmp1的启动子或者Dspp的mRNA上调节成牙本质向分化。以上结果为今后成牙本质细胞分化miRNA调控网络的构建打下了实验基础，并加深了对调控成牙本质细胞分化的分子机制的理解。