靶向抑制脯氨酰羟化酶2通过HIF-1α调控miR-421在椎间盘退变中的作用及机制研究

Degenerative disc disease is a common cause of chronic back pain, which is the leading global cause of disability in most countries. Disc degeneration is characterized by apoptosis of nucleus pulposus (NP) cell and degradation of extracellular matrix. PHD2 is the critical oxygen sensor regulating oxygen-dependent degradation of hypoxia inducible factor (HIF-1α). HIF-1α, when stabilized by hypoxic condition, drives a transcriptional programme that promotes hypoxia adaptation, and has important roles in ischaemic and inflammatory diseases. Our previous work showed that silencing of PHD2 in NP cells significantly decreased TNF-α-induced expression of inflammatory cytokines, including Fas, FasL, MMP-3, -13, SDC4, COX2, while partially restoring aggrecan and collagen II expression. PHD2 may offer an attractive therapeutic target to treat catabolic events in the pathogenesis of intervertebral disc disease. However, the function of PHD2 in NP apoptosis is still unknown. Growing evidence shows that microRNA (miRNA) can regulate many aspects of NP cell activity, which may bring about novel drug candidates and biomarkers. Since the NP resides in a unique hypoxic niche, research on hypoxia-mediated miRNA is of great significance. A recent report claimed that miR-421 regulated by HIF-1α inhibits apoptosis and promotes proliferation in gastric cancer. We speculate that inhibition of PHD2 can induce miR-421 expression through HIF-1α activation and thus suppress apoptosis of NP cells. Based on preliminary results, this project will aim to explore whether miR-421 modulated by HIF-1α suppresses NP apoptosis through Fas/FasL pathway, and elucidate the mechanisms of PHD2/HIF-1α/miR-421 signaling in NP apoptosis. Based on a self-designed intervertebral disc degeneration model in rat, we will explore if targeted therapy on blocking the PHD2/HIF-1α/miR-421 signaling may be a therapeutic target for disc degeneration through inhibition of apoptosis, suppression of inflammation as well as accumulation of extracellular matrix.

椎间盘退变疾病临床极为常见，髓核细胞凋亡是其重要的病理基础。项目组前期研究表明，脯氨酰羟化酶2（PHD2）是髓核细胞内降解缺氧诱导因子HIF-1α的关键酶；在髓核细胞中抑制PHD2不仅具有抗炎和促基质合成作用，还能减少凋亡因子Fas、FasL表达。然而，PHD2在髓核细胞凋亡中的作用机制仍不清楚。最新研究表明，肿瘤细胞中的miR-421受HIF-1α调控，具有抗凋亡作用。我们推测，抑制髓核细胞中的PHD2能通过激活HIF-1α，上调miR-421表达，从而减少细胞凋亡。本课题将结合前期结果，研究髓核细胞中的miR-421是否受HIF-1α调控，及其对Fas/FasL凋亡途径的作用；阐明PHD2/HIF-1α/miR-421信号通路在髓核细胞凋亡中的作用机制；在大鼠椎间盘退变模型中研究以PHD2/HIF-1α/miR-421信号通路为靶向的治疗效果，为探索椎间盘退变的治疗手段提供新的思路。

脯氨酰羟化酶2(PHD2)是细胞内重要的氧感受器，是调控缺氧诱导因子降解的关键酶。抑制PHD2可以在多种细胞发挥抗炎、抗缺血、改善免疫功能等作用。在本研究中，我们通过体外通过信号通路阻滞剂、siRNA沉默技术及基因转染技术发现，过表达miR-421能够减少髓核细胞表达凋亡因子Fas、FasL，从而抑制细胞凋亡。而抑制miR-421并不影响细胞凋亡。髓核细胞内的miR-421受氧含量调控，抑制PHD2能通过激活HIF-1α，上调miR-421表达，从而减少细胞凋亡。在此基础上，于已创建的椎间盘退变模型中，验证了以抑制PHD2为靶向的治疗能够减少髓核细胞凋亡，减缓细胞外基质破坏，从而减缓椎间盘退变，而在椎间盘内过表达miR-421未能改变椎间盘退变的病理过程。结合我们的前期工作基础，以抑制PHD2为靶向的治疗，能够通过激活HIF-1α信号通路，抑制NF-κB信号通路，减少髓核细胞凋亡，促进细胞外基质合成，减轻椎间盘退变程度。而作为众多miRNA分子中的一员，miR-421分子在髓核细胞中出凋亡以外的生物过程中作用还有待进一步研究。此外，小分子非编码 RNA 在细胞细胞增殖分化和凋亡，血管生成以及炎症反应等生物活动中调控作用是近年来的研究热点。通过高通量测序技术，本研究发现椎间盘损伤后，椎间盘内存在大量转运 RNA（tRNA） 来源的小片段（tRNA-derived small RNAs， tsRNA）改变， 并在髓核细胞中得到初步验证，因此，tsRNA可能为椎间盘退变机制研究提供新的思路。