miR-145靶向作用Sox9调控TGF-β信号通路参与下颌髁突软骨生长发育的机制研究

TGF-β/Smads/Sox9 signaling pathway plays a critical role in regulating condylar cartilage growth and development, the precise mechanisms of which remain to be defined. It has been reported recently that miR-145 is a novel regulator at early stage of chondrogenic differentiation of the mesenchymal stem cells by directly targeting Sox9. Our study found that miR-145 expression showed fluctuating changes in response to TGF-β2 stimulation, which suggesting miR-145 may acts as a key mediator of TGF-β signaling pathway in condylar cartilage growth and development. The purpose of this study is to observe the temporal spatial expression of miR-145 and Sox9 in the growth and development of the mandibular condylar cartilage through animal model in vivo. The research also includes investigation of the influence of TGF-β2 on the miR-145 and Sox9 expression via establishing mandibular condylar cartilage and chondrocytes culture model in vitro. Furthermore, Dual-luciferase reporter gene assay is utilized for the validation of the putative binding site of miR-145 targeting Sox9. Finally, the influence of over expression or inhibition of miR-145 on the Sox9 and other chondrogenic markers expression as well as the TGF-β/Smads signaling pathway is explored by the application of cell transfection assay. In conclusion, the objective of the study is to dissect the effects and molecular mechanisms of modulation of the TGF-β signal transduction pathway through direct targeting Sox9 by miR-145, thus providing the experimental evidence for deep insight into the underlying mechanisms of the condylar cartilage growth and development.

TGF-β/Smads/Sox9信号通路在下颌髁突软骨的生长发育中具有重要作用，但其中的调控机制还未被完全阐明。近来的研究表明，miR-145能够通过Sox9介导调控间充质干细胞的成软骨向分化。本课题组发现，髁突软骨细胞内miR-145的表达变化与TGF-β2的刺激有关，推测miR-145可能通过作用TGF-β通路调控髁突软骨的生长发育。本课题旨在通过动物模型观察miR-145和Sox9在髁突软骨生长发育中的时空表达特点；建立髁突软骨细胞及软骨培养模型，探讨TGF-β2对miR-145及Sox9表达的影响；应用荧光素酶报告基因系统检测miR-145与Sox9的作用位点；最后运用细胞转染技术，明确增强或抑制miR-145的表达对Sox9以及TGF-β信号通路转导的影响。通过研究揭示miR-145靶向作用Sox9调控TGF-β信号通路的机制，以期为阐明髁突软骨生长发育的调控机理提供实验依据。

非编码RNA在调控软骨生长发育、软骨细胞增殖、分化肥大、炎症微环境下的内稳态方面发挥关键作用。其中，长链非编码RNA（lncRNAs）与微小RNA（microRNAs）通过TGF-β/Smads/Sox9信号通路调控软骨细胞的生长分化以及炎症反应的分子机制还不甚清楚。本课题建立了小鼠ATDC5细胞的体外成软骨分化模型，运用高通量的基因芯片技术，检测软骨细胞分化过程中lncRNAs的差异表达情况，新发现了lncRNA AK136902在软骨细胞分化过程中表达显著上调，且与软骨分化重要转录因子Sox9以及软骨分化标记分子Aggrecan的表达高度相关。体内实验也发现，lncRNA AK136902在C57BL/6小鼠的纤维软骨以及透明软骨中具有明显的时空表达特异性，可以作为软骨细胞分化成熟的一个新的参考指标。后续的功能实验发现，lncRNA AK136902可以通过Sox9来调控软骨分化标记分子Aggrecan的表达。此外，我们还通过IL-1β和TNF-α联合刺激建立了SD大鼠髁突软骨细胞的体外炎症模型，检测了髁突软骨细胞炎症微环境中miRNAs的差异表达情况，发现miRNA-146a-5p在炎症髁突软骨细胞中高表达。通过生物信息分析、双荧光素酶实验发现ox40l是miRNA-146a-5p的靶基因。后续的功能实验发现，miR-146a-5p在髁突软骨细胞的炎症微环境中可作为一种非专业抗原递呈细胞，通过ox40l影响T细胞介导的免疫功能。本研究从一个新的角度探索髁突软骨细胞分化以及炎症反应的的分子调控机制，为揭示髁突软骨生长发育机制以及软骨疾病的防治提供实验依据。