CircRNA-microRNA相互对话调控GDF5/SIRT6信号通路在椎间盘退行性变中的作用及其机制研究

Low back pain resulted from intervertebral disc degeneration (IDD) brings heavy burden to the society. Previous studies confirmed that IDD demonstrated GDF5/SIRT6 pathway being excessively inhibited and this was found to be associated with abnormally high expression of microRNA(miR-34a). Recent studies have shown that circRNA-miRNA mutual regulation and dynamic balance disorder plays an important role in the development of many diseases. But there are very few reports on researches about functions of circRNA in IDD. In the early stage, we discovered a new circRNA targeting miR-34a (which is temporarily called ciRS-34a) in the nucleus pulposus by using high-throughput sequencing and bioinformatics. This study was to investigate whether ciRS-34a-miR-34a mutual regulation will result in GDF5 pathway inhibition and then lead to IDD by using clinical samples, cell experiments, animal models, as well as RNA-FISH and other technologies. And we used specificity ways to change the ciRS-34a expressions, and tried to remit and reverse IDD to make further research on the effects of ciRS-34a-miR-34a mutual regulations for GDF5/SIRT6 pathway inhibition in IDD pathogenesis, so as to provide new thoughts for IDD early clinical diagnosis and treatments.

椎间盘退变（IDD）导致的下腰痛给社会带来沉重负担。前期研究证实IDD表现出GDF5/SIRT6通路过度抑制并发现其与异常高表达的微小RNA（miR-34a）有关。近年研究表明环状RNA（circRNA）-miRNA相互调控和动态平衡的紊乱在众多疾病发展中有重要作用，但IDD中环状RNA功能研究鲜有报道。我们前期通过高通量测序和生物信息学在髓核组织中发现一个新的以miR-34a为靶点的circRNA，暂称ciRS-34a。本课题拟通过临床标本、细胞实验、动物模型层面，采用RNA-FISH等技术，明确ciRS-34a-miR-34a相互调控，是否引起GDF5通路抑制，继而导致IDD，并运用特异性手段改变ciRS-34a表达，尝试缓解和逆转IDD，从而深入探讨ciRS-34a-miR-34a相互调控抑制GDF5/SIRT6通路在IDD发病机制中的作用,为IDD的临床早期诊断和治疗提供新的思路。

椎间盘退变（IDD）导致的下腰痛是全世界最常见的老年慢性致残性疾病之一。本课题组主要研究了早期椎间盘退变发生发展过程中SIRT蛋白的过度抑制介导的髓核细胞功能学改变及其精确的分子机制。我们前期研究发现了介导SIRT通路抑制的miR-34a。环状RNA(CircRNA)是一类新型的内源性非编码RNA(NcRNA)，可以作为miRNA的分子海绵从而在不同疾病的病理生理中发挥作用。研究发现，circRNA在退变椎间盘中呈现显著差异性表达，提示其与椎间盘生理病理过程有着非常密切的关系。在本项目的执行过程中，我们通过对压力处理后的髓核细胞进行测序，发现了和髓核细胞损伤密切相关的一种circRNA，我们将其确认为circRNA-CIDN，其在压力引起的退变髓核细胞中显著下调。通过生物信息学方法进行circRNA的靶基因预测，我们发现circRNA-CIDN可以作为miR-34a-5p的分子海绵，可能在SIRT1信号通路中起重要的调控作用。进一步的病例样本检测，我们发现miR-34a-5p在退变椎间盘髓核细胞中表达明显升高，同时circRNA-CIDN和SIRT1蛋白的表达明显下调。我们通过荧光素酶和RNA免疫沉淀(RIP)实验观察到circRNA-CIDN作为miRNA海绵与miRNAs结合，并证实了circRNA-CIDN能够直接靶向调控miR-34a-5p和SIRT1的表达。通过构建circRNA-CIDN的载体和microRNA模拟物，我们发现过表达circRNA-CIDN显著抑制了miR-34a-5p的功能，并提高了SIRT1的表达水平，从而缓解了压力诱导的髓核细胞凋亡和细胞外基质降解。体内研究表明，circRNA-CIDN的椎间盘过表达提高了椎间盘中SIRT1, Bcl-2，aggrecan, collagen II的蛋白水平，同时抑制了MMP-3, MMP-13 和Bax 的表达，继而减轻了椎间盘髓核细胞在压力模型下的退变表型和IDD的发生。本研究表明，运用特异性干预手段，改变SIRT 通路相关的miRNA和circRNA水平，可以缓解或逆转IDD的发展，为IDD的临床早期诊断和治疗提供新的思路。本课题已发表5分以上SCI论文4篇，10分以上SCI论文1篇，培养硕士生3名。