circ_0072746靶向miR-29介导自噬和成骨基因信号通路参与调控脊柱软骨终板退变钙化

Disc degeneration diseases include cervical spondylosis and lumbar disc herniation, the incidence of which has increased year by year. The degeneration of endplate car-tilage is considered as the initial factor of disc degeneration. At present, the molecu-lar mechanism of endplate cartilage degeneration is not clear. Applicants found that miR-29 is over-expression in endplate cartilage of cervical spine, and overexpres-sion of miR-29 in vitro induces degeneration and calcification of endplate chondro-cytes. Further study found that miR-29 activates mTORC1 signaling pathway through PTEN to play its biological role. However, it is unclear whether activates mTORC1 is involved in the regulation of degeneration and calcification of endplate cartilage through the downstream ULK1 autophagy signaling pathway or S6K1/eIF4E-BP1 osteogenic gene signaling pathway or co-role. More importantly, the research group confirmed that circRNA is low expression in the degenerative calcified spinal endplate cartilage by Genechip and qPCR, and the target relationship between the circRNA and miR-29 was confirmed by the technology of luciferase. Hence, we propose hypothesis that circRNA targeting miR-29 mediate the PTEN-mTORC1-ULK1 autophagy signaling pathway and the mTORC1-S6K1/ eIF4E-BP1 osteogenic signal pathway to regulate spinal endplate cartilage degenera-tion. Through molecular biology techniques including gene silencing and overex-pression, we attempt to reveal the mechanism of circRNA in the regulation of spinal endplate cartilage degeneration in vitro and in vivo experiments in order to provide new ideas for the target treatment of degenerative disc disease in the future.

椎间盘退变性疾病发病率逐年增加，软骨终板退变是椎间盘退变的始动因素。目前软骨终板退变的分子机制不清。申请人发现miR-29在退变的颈椎软骨终板中高表达，体外过表达miR-29诱发软骨细胞发生退变钙化。进一步研究发现miR-29通过PTEN激活mTORC1信号通路发挥生物学作用。然而，激活的mTORC1是通过下游的ULK1自噬信号通路，还是通过S6K1/eIF4E-BP1成骨基因信号通路参与调控终板软骨退变钙化，尚不明确。更为重要的是，课题组基因芯片和qPCR实验证实circ_0072746在软骨终板退变组中低表达，荧光素酶实验验证了其与miR-29靶向关系。提出假说：circ_0072746靶向miR-29介导自噬信号通路和成骨基因信号通路参与调控脊柱软骨终板退变。我们拟通过分子生物学技术，体内外实验揭示环状RNA参与调控脊柱软骨终板退变的作用机制，为日后通过靶点治疗椎间盘退变提供新思路。

椎间盘退变是多种脊柱疾病的重要病理基础，其引起的椎间盘突出、椎管狭窄以及椎体滑脱等疾病在临床上常见，严重影响患者生活质量。软骨终板退变是椎间盘退变的始动因素。目前软骨终板退变的分子机制不清。circRNA作为竞争性内源RNA在调控疾病发生发展过程中起重要作用。我们前期发现miR-29在退变的颈椎软骨终板中高表达，体外过表达miR-29诱发软骨细胞发生退变钙化。进一步研究发现miR-29通过PTEN激活mTORC1信号通路发挥生物学作用。然而，激活的mTORC1是通过下游的ULK1自噬信号通路，还是通过S6K1/eIF4E-BP1成骨基因信号通路参与调控终板软骨退变钙化，尚不明确。通过基因芯片和qPCR实验，我们证实circ_0072746在软骨终板退变组中低表达，荧光素酶实验验证了其与miR-29靶向关系。该项目应用qRT-PCR 和 Western blot、免疫荧光、FISH等技术分析了circ_0072746及其下游效应因子miR-29的功能与靶向关系，通过体外过表达及敲低实验，证实其通过介导PTEN激活mTORC1-ULK1自噬信号通路及mTOR-S6K1/eIF4E- BP1成骨基因信号通路参与调控软骨终板退变钙化的生物学行为，为日后通过靶点治疗椎间盘退变提供新思路。