低氧诱导的ciRS-7/miR-7通过p38信号通路调控牙周膜干细胞成骨分化的机制

The imbalance between "hypoxia-induced osteogenesis" and " inflammation-induced anti-osteogenesis" is essential to aggravate the progression of periodontal disease. PARP1/p38 signalling pathway can induce the osteogenic differentiation, yet the understanding of the molecular mechanisms controlling osteogenic differentiation of periodontal ligament stem cells(PDLSCs) remains poor. Our previous study showed that the expression levels of ciRS-7 and miR-7 were significantly increased in PDLSCs treated by hypoxia, and the target relationship between miR-7 and PARP1 was predicted using bioinformatics. Therefore, we propose a hypothesis: hypoxia-induced ciRS-7/miR-7-PARP1-p38 signalling pathway stimulates osteogenic differentiation of PDLSCs. In present study, cellular experiments will be performed to evaluate the regulatory mechanism among ciRS-7, miR-7, PARP1. We further explore that ciRS-7 by RNA interference whether release miR-7 to suppress the expression of PARP1 under hypoxia. Finally, we analyse the effects of hypoxia-induced ciRS-7/miR-7-PARP1-p38 signalling pathway on osteogenic differentiation of PDLSCs via in vivo. This project may provide new clues for hypoxia-induced osteogenic differentiation of PDLSCs to reconstruct the periodontal tissue.

“低氧→成骨”与“炎症→抑骨”失衡是促使牙周病变不断进展的关键因素，PARP1/p38信号通路可诱导成骨分化，但是其在牙周膜干细胞成骨分化过程中的作用机制尚不清晰。我们前期研究发现牙周膜干细胞经低氧处理后ciRS-7和miR-7均表达升高，同时生物信息学分析表明PARP1是miR-7的靶基因，由此提出假设：低氧通过诱导ciRS-7/miR-7-PARP1-p38信号通路促进牙周膜干细胞的成骨分化。本课题拟通过多种细胞学实验评价ciRS-7-miR-7-PARP1三者在牙周膜干细胞中的靶向调控关系；低氧环境下干扰ciRS-7的表达，是否能够解除ciRS-7对miR-7的吸附富集，促进miR-7对PARP1的靶向抑制；同时结合体内实验进一步研究低氧环境下ciRS-7-miR-7-PARP1-p38信号通路对牙周膜干细胞成骨分化的影响，为临床牙周组织的修复重建提供新的分子靶点。

牙周膜干细胞具有自我更新和多向分化的特性。内质网应激/自噬是在缺氧、炎症和癌症等多种微环境下诱导的重要细胞过程。环状RNAs在缺氧条件下对某些疾病有重要的调节作用，但在缺氧条件下PDLSCs中的ciRs-7在牙周炎中ciRs-8对内质网应激/自噬的调节作用尚未见报道。microRNA-7（miR-7）是最早发现的microRNAs之一，通过抑制靶基因的表达参与多种生物学过程，包括细胞增殖、凋亡、细胞周期和成骨分化，但miR-7在牙周膜干细胞中是如何调控生物学过程的，目前尚不清楚。牙周炎是一种慢性感染性口腔疾病，是一种牙槽骨丢失、血管异常的牙齿支持组织疾病。炎性浸润相关的血管数目和直径，导致牙周组织缺血缺氧。在本研究中，我们证实了低氧和miR-7对牙周膜干细胞内质网应激/自噬中的调节作用，并进一步研究了在缺氧微环境下如何通过激活内质网应激/自噬抑制成骨分化和诱导凋亡。我们的数据表明，短期缺氧通过核内RUNX2积聚促进PDLSC成骨，而长期缺氧则抑制成骨。此外，缺氧通过升高内质网应激/自噬增加了促进了成骨分化。miR-7过表达诱导PDLSCs内质网应激/自噬和凋亡，降低分化。此外，自噬抑制剂3-MA逆转了CoCl2刺激对PDLSCs成骨的抑制作用。在缺氧微环境中，miR-7通过诱导内质网应激/自噬共同调节PDLSCs的成骨分化和凋亡。