YAP通过miR-17家族调控p21的表达影响OA软骨表型衰老的机制研究

The senescence of chondrocyte plays a canonical role in cartilage degenerative diseases, mainly osteoarthritis (OA). However, the molecular mechanism underlying its senescence is largely illusive. Several studies have shown that YAP negatively regulates the differentiation and maturation of chondrocyte, and is also involved in modulating the senescence of multiple cell types. Moreover, during our recent experiments we came up with the following important findings, in which cytoplasmic YAP maintains the phenotype of cartilage-associated cells; OA chondrocyte exhibits prominent characteristics of senescence, while the knockdown or overexpression of YAP and its point-mutated mutants confirms that the expression of p21 is negatively regulated by YAP; In contrast, high-throughput miRNAs sequencing reveals that YAP is in control of the biosynthesis of a series of important miRNAs including senescence relevant miR-17 family. Based on these findings, we then hypothesized that YAP mediates the phenotypic senescence and degeneration of OA cartilage via targeting the miR-17 family regulated p21 expression. Therefore, the present study intends to provide a clear evidence in order to support the above hypothesis at different levels including clinical specimens, cell phenotypes, molecular levels, and animal models. In addition, YAP-conditional-knockout mice are used to demonstrate the biological function and significance of YAP-mediated miR-17/p21 axis in the phenotypic senescence of OA cartilage. Collectively, The finding of which, YAP/miR-17/p21 signaling plays a crucial role in regulating chondrocyte senescence sheds light upon the illusive role of senescence regarding the degenerative mechanism of OA cartilage, and provides a new theoretical and experimental basis for future research and development of senescence-based OA drugs.

软骨细胞的衰老在OA软骨退变中起关键作用，然而其分子机制仍不明确。已知YAP负调控软骨细胞分化及成熟，且参与调控多种细胞的衰老。我们最近研究发现：细胞质中YAP维持软骨相关细胞的表型；OA患者软骨细胞具有明显衰老特征，采用敲低、过表达YAP及其突变体明确其机制与YAP负调控p21的表达有关；高通量转录组测序发现YAP调控一系列包括衰老相关miR-17在内重要miRNAs的生物合成。由此推测YAP通过调控miR-17家族成员靶向p21的表达，从而介导OA软骨细胞衰老及软骨退变。本研究拟从临床组织、细胞表型、分子水平及动物模型等多个层面证明之，并利用YAP条件性基因敲除小鼠从体内证实YAP通过miR-17/p21影响OA软骨表型衰老的关键作用及意义。本项目有望以细胞衰老及YAP/miR-17/p21信号轴为视角，为阐明OA软骨退变的衰老机制及基于衰老防治OA药物的研发提供新理论及实验依据。

软骨细胞的衰老在OA软骨退变中起关键作用，然而其分子机制仍不明确。已知YAP负调控软骨细胞分化及成熟，且参与调控多种细胞的衰老。我们最近研究发现：细胞质中YAP维持软骨相关细胞的表型；OA患者软骨细胞具有明显衰老特征，采用敲低、过表达YAP及其突变体明确其机制与YAP负调控p21的表达有关；高通量转录组测序发现YAP调控一系列包括衰老相关miR-17在内重要miRNAs的生物合成。由此推测YAP通过调控miR-17家族成员靶向p21的表达，从而介导OA软骨细胞衰老及软骨退变。本研究从临床组织、细胞表型、分子水平及动物模型等多个层面证实YAP通过miR-17/p21影响OA软骨表型衰老的关键作用及意义。项目以细胞衰老及YAP/miR-17/p21信号轴为视角，阐明OA软骨退变的衰老机制及基于衰老防治OA药物的研发提供新理论及实验依据。按照项目计划，我们着重探讨YAP通过靶向过miR-17家族，尤其是miR-93-5p，调节p21的表达，进而调控OA软骨的“衰老”。目前项目已按照计划完成对YAP上下游信号通路及具体分子机制作了深入的探索，我们发现在OA动物模型及OA临床样本中YAP可能与OA的病程相关，体外实验证实YAP的亚细胞定位负调控P21影响细胞衰老表型，miR-17家族，尤其是miR-93-5p参与这一过程。此外，我通过临床样本单细胞测序分析发现OA及DOA滑膜中存在M1巨噬细胞浸润，动物模型也验证了这一发现，体外研究发现滑膜细胞糖酵解增强了巨噬细胞的募集和活化，且YAP是参与这过程的关键蛋白。以上研究发现为阐明OA软骨“衰老”的机制及其靶向治疗提供新的线索及实验依据。以上研究发现为阐明OA软骨“衰老”的机制及其靶向治疗提供新的线索及实验依据。