应力作用下miR-142-3p调控YAP/TAZ通路促进软骨终板干细胞成骨分化的机制研究

The incidence of degenerative disc disease (DDD) is high, the mechanism is unknown, and it is a serious hazard to human health. Studies have shown that cartilage endplates are the most important nutrient pathways for intervertebral discs, and stress-induced abnormal calcification of cartilage endplates is the initiating factor of degenerative disc disease. We found that the osteogenic differentiation of endplate stem cells (CESCs) under intermittent stress may be the cause of endplate calcification, but the specific mechanism is still unclear. Previous studies have found that under the intermittent cyclic stress stimulation, RUNX2 and other osteogenic gene expression increased in CESCs, while several mechanical signal transduction pathway-related microRNAs such as miR-142-3p were upregulated. Therefore, we hypothesized that in the endplate of intervertebral disc, CECSs up-regulated the expression of miRNA under intermittent stress, inhibiting the expression of Lats1, promoting the activation of YAP/TAZ into the nucleus, activating the expression of downstream RUNX 2, and promoting the osteogenic differentiation of CECSs，and resulting in endplate calcification.We would use CECSs, animal and clinical specimens as materials, using RT-PCR, Western blot, immunohistochemistry,immunofluorescence, co-immunoprecipitation and other experimental methods to explore the regulation mechanism of miR-142-3p-YAP/TAZ pathway under stress to promote CESCs osteogenic differentiation and endplate calcification.

椎间盘退变性疾病（DDD）发病率高，机制不明，严重危害人类健康。软骨终板是椎间盘最重要的营养途径，应力作用下，软骨终板干细胞（CESCs）成骨分化导致软骨终板异常钙化是椎间盘退变性疾病的始动因素，但具体机制尚不清楚。课题组前期研究发现，CESCs在间歇循环应力刺激下， 成骨基因RUNX2等表达升高，同时miR-142-3p显著上调，并可能通过力学信号相关YAP/TAZ通路促使CESCs成骨分化。因此我们假设，间歇应力作用下，力感应miR-142-3p表达上调，通过抑制Lats1表达，促使YAP/TAZ活化入核，激活下游RUNX2表达，进而促使CECSs成骨分化。本项目拟用CECSs、动物及临床标本为材料，采用RT-PCR、Western blot、免疫组化、免疫荧光、免疫共沉淀等实验方法，探讨应力作用下Hippo-YAP/TAZ通路调节终板钙化的作用机制。

椎间盘退变性疾病(DDD)发病率高，机制不明，严重危害人类健康。软骨终板是椎间盘最重要的营养途径，应力作用下，软骨终板干细胞(CESCs)成骨分化导致软骨终板异常钙化是椎间盘退变性疾病的始动因素，但具体机制尚不清楚。课题组前期研究发现，CESCs在间歇循环应力刺激下，成骨基因RUNX2等表达升高，同时miR-142-3p显著上调，并可能通过力学信号相关YAP/TAZ通路促使CESCs成骨分化。我们对CESCs 进行间歇循环应力(ICMT)刺激，以及转染miR-142-39 siRNA干扰，发现CESCs的RUNX2、Col 1 等成骨标记物mRNA 及蛋白表达升高，证实调控CE SCs 成骨分化的关键miRNA为miR-142-3p。随之我们对CESCs 进行间歇循环应力(ICMT)刺激，以及转染lats1 siRNA干扰，发现CESCs的RUNX2、Col 1等成骨标记物mRNA 及蛋白表达升高，证明miR-1 42-3p通过LATS1调控CESCs 成骨分化。我们的研究证实了椎间盘终板中， CECSs在应力作用下，miR-142-3p表达上调，通过抑制Lats1表达，促使 YAP/TAZ活化入核，激活下游RUNX2表达，进而促使CECSs成骨分化，进而引起终板钙化。我们的研究一方面丰富了椎间盘退变的机制研究内容，另一方面为其干预提供新的干预靶点
。