Wang Zuobo, Sun Lin, Jia Kaixue, Wang Hongxia, Wang Xiuxiang
Rehabitation Department, Yantai Hospital of Traditional Chinese Medicine, 264001, China.
Department of Neurology, Yantai Hospital of Traditional Chinese Medicine, 264001, China.
Neurosci Lett. 2019 May 14;701:226-233. doi: 10.1016/j.neulet.2019.02.038. Epub 2019 Feb 28.
Parkinson's disease (PD) is a most common progressive neurodegenerative disease mainly occurring in the elderly. Plenty of miRNAs are reported to involve in the progression of PD. However, the role of miR-9-5p in the regulation of PD pathogenesis remains unclear. The expressions of miR-9-5p and Sirtuin 1 (SIRT1) at mRNA and protein levels were determined by qRT-PCR and western blotting (WB) analyses. Cell viability and apoptosis were evaluated by MTT and flow cytometry. The levels of apoptosis-related proteins Bcl-2, Bax, Caspase-3 were detected by WB analysis. The releases of inflammatory cytokines IL-1β and TNF-α were examined by ELISA assay. ROS generation, LDH and SOD activity were evaluated using commercially available kits. Bioinformatics analysis, luciferase reporter, and qRT-PCR assays were performed to demonstrate the true interaction between miR-9-5p and SIRT1. Results showed miR-9-5p was upregulated and SIRT1 was downregulated in MPP-treated SH-SY5Y cells in dose- and time- dependent manners. miR-9-5p knockdown attenuated MPP-induced neurotoxicity in SH-SY5Y cells, as evidenced by the enhancement in cell viability, and the suppression in cell apoptosis, inflammation, and oxidative stress. SIRT1 was identified to be a target of miR-9-5p. Restoration of miR-9-5p aggravated SIRT1-attenuated neurotoxicity in MPP-treated SH-SY5Y cells. Our data suggested these data indicated that miR-9-5p exerted a neurotoxic role in MPP-derived PD by directly targeting STAT1, providing a potential therapeutic strategy for patients troubled by PD.
帕金森病（PD）是一种最常见的主要发生于老年人的进行性神经退行性疾病。大量的微小RNA（miRNA）被报道参与了帕金森病的进展。然而，miR-9-5p在帕金森病发病机制调控中的作用仍不清楚。通过定量逆转录聚合酶链反应（qRT-PCR）和蛋白质免疫印迹（WB）分析来测定miR-9-5p和沉默调节蛋白1（SIRT1）在mRNA和蛋白质水平的表达。通过MTT法和流式细胞术评估细胞活力和细胞凋亡情况。通过WB分析检测凋亡相关蛋白Bcl-2、Bax、半胱天冬酶-3的水平。通过酶联免疫吸附测定（ELISA）检测炎性细胞因子白细胞介素-1β（IL-1β）和肿瘤坏死因子-α（TNF-α）的释放。使用商用试剂盒评估活性氧（ROS）生成、乳酸脱氢酶（LDH）和超氧化物歧化酶（SOD）活性。进行生物信息学分析、荧光素酶报告基因检测和qRT-PCR检测以证实miR-9-5p与SIRT1之间的真实相互作用。结果显示，在1-甲基-4-苯基吡啶离子（MPP）处理的SH-SY5Y细胞中，miR-9-5p呈剂量和时间依赖性上调，而SIRT1呈剂量和时间依赖性下调。敲低miR-9-5p可减轻MPP诱导的SH-SY5Y细胞神经毒性，表现为细胞活力增强，细胞凋亡、炎症和氧化应激受到抑制。SIRT1被确定为miR-9-5p的一个靶点。恢复miR-9-5p可加重SIRT1减轻的MPP处理的SH-SY5Y细胞的神经毒性。我们的数据表明，这些数据表明miR-9-5p通过直接靶向信号转导和转录激活因子1（STAT1）在MPP诱导的帕金森病中发挥神经毒性作用，为受帕金森病困扰的患者提供了一种潜在的治疗策略。
