miR-4639-5p的表达调控及其异常在帕金森病发病中作用的研究

DJ-1 protein protects cells against oxidative stress. DJ-1 deficiency plays a crucial role in oxidative insult and Parkinson's disease (PD) pathogenesis. In familial PD, mutations of DJ-1 gene can cause instability of the protein which makes DJ-1 easily degraded by proteasome. Reduced DJ-1 protein level has also been reported in substantia nigra of sporadic PD patients, but the mechanism is unclear. Our previous study showed that DJ-1 gene is post-transcriptionally regulated by hsa-miR-4639-5p. Plasma miR-4639-5p level was identified to be significantly up-regulated in PD patients compared to healthy controls. Moreover, abnormal up-regulated plasma miR-4639-5p was derived from exosomes, which reflects pathological changes in the central nervous system, relating to abnormal decreased DJ-1 protein level, but the underlying mechanism is still obscure. In this proposal, we are going to identify the promoter region and the regulatory elements of hsa-miR-4639-5p. Based on the results, we will continuously investigate the abnormal regulation of hsa-miR-4639-5p expression in pathological conditions of PD. All these will help us to understand the molecular mechanism underlying DJ-1 deficiency, burst of oxidative stress and neuronal death in PD.

DJ-1可保护细胞抵抗氧化应激的攻击，其下降被认为与氧化应激损伤和帕金森病（PD）发病有关。家族性PD患者中DJ-1基因突变可导致DJ-1易被蛋白酶体所降解，散发性PD患者中黑质部DJ-1也有下降，但机制不明，了解其异常机制可帮助阐明散发性PD患者的发病机制。我们前期研究发现hsa-miR-4639-5p可调控DJ-1的表达， PD患者外周血中hsa-miR-4639-5p水平较正常对照显著升高，且其异常增高主要来源于中枢神经系统的改变，与DJ-1水平下降有关，但hsa-miR-4639-5p异常上调的原因仍不明确。我们将从miRNA的表达调控入手，通过研究明确hsa-miR-4639-5p的启动子和上游调节因子，在此基础上观察PD病理状态下hsa-miR-4639-5p调控异常与DJ-1水平异常降低、氧化应激异常增加和多巴胺能神经元变性死亡间的关系，以期帮助阐释散发性PD发生的分子机制。

DJ-1蛋白为细胞内重要的抗氧化应激因子，其水平下降可导致神经元遭受氧化应激损伤，与帕金森病（PD）的发生发展密切相关。我们前期研究发现，microRNA可调控DJ-1表达，并进一步发现hsa-miR-4639-5p为人DJ-1蛋白的特异性转录后调节因子。hsa-miR-4639-5p的上调可导致DJ-1水平降低、氧化应激损伤和神经元死亡。此外，PD患者血浆中hsa-miR-4639-5p水平显著上调，且其异常增高主要来源于中枢神经系统的改变，而这可能导致了中枢神经系统中DJ-1水平的下降。目前，hsa-miR-4639-5p异常上调的原因仍不明确。探索hsa-miR-4639-5p异常上调的机制将有助于阐释PD中DJ-1水平异常降低、氧化应激异常增加和多巴胺能神经元变性死亡的原因，以期帮助阐释散发性PD发生的分子机制。本项目从miRNA的表达调控入手，探索了hsa-miR-4639-5p异常上调的表达调控机制。通过双荧光素酶报告基因实验及CRISPR-Cas9基因定向敲除技术鉴定出调控hsa-miR-4639-5p转录的上游核心启动子区域；在对上游调节因子的筛选中，通过检测PD患者DNA甲基化水平检测及进一步使用DNA甲基转移酶抑制剂验证，发现DNA甲基化不参与hsa-miR-4639-5p的表达调控；通过使用泛去乙酰化酶抑制剂发现hsa-miR-4639-5p的表达受到组蛋白乙酰化修饰水平的调控，进一步通过特异性去乙酰化酶抑制剂、过表达及敲减实验明确去乙酰化酶HDAC11为调控hsa-miR-4639-5p表达的关键因子，HDAC11水平的改变可通过影响hsa-miR-4639-5p的转录，影响hsa-miR-4639-5p的表达水平、DJ-1蛋白水平及细胞内氧化应激水平；此外，发现hsa-miR-4639-5p启动子区单核苷酸多态性（SNP）与hsa-miR-4639-5p 表达水平和PD风险密切相关。该项目的研究成果为我们前期所发现的hsa-miR-4639-5p可作为PD早期诊断标志物提供了更多的证据，有助于加深对散发性PD发病机制的认识，为PD的干预治疗及药物靶点提供新的理论和实验依据。