Song Yanfeng, Liu Ying, Chen Xiaowei
Department of Internal Medicine-Neurology, Hua Mei Branch of the Second People's Hospital of Liaocheng, Linqing, China.
Yonsei Med J. 2018 May;59(3):416-424. doi: 10.3349/ymj.2018.59.3.416.
Parkinson's disease (PD) is a common age-dependent neurodegenerative disease. MiR-212 has been demonstrated to exert protective effects in several neurological disorders. The present study aimed to investigate the role and underlying molecular mechanism of miR-212 in PD.
1-methyl-4-phenylpyridinium (MPP+)-induced SH-SY5Y cells were applied as a PD model in vitro. RT-qPCR was used to measure the expression of miR-212 and Kruppel-like factor 4 (KLF4) mRNA. Western blot analysis was performed to detect the protein levels of KLF4, Notch1 and Jagged1. Cell viability and apoptosis were determined by the Cell Counting Kit-8 and flow cytometry, respectively. Quantitative analysis of caspase-3 activity, lactate dehydrogenase (LDH), reactive oxygen species (ROS), superoxide dismutase (SOD), tumor necrosis factor-α (TNF-α), and interleukin-1 beta (IL-1β) was conducted with corresponding ELISA kits. Dual-luciferase reporter assay was employed to evaluate the relationship between miR-212 and KLF4.
MiR-212 was downregulated in MPP⁺-induced SH-SY5Y cells. Also, miR-212 alleviated MPP⁺-induced SH-SY5Y cell damage, embodied by increased cell viability, decreased caspase-3 activity, LDH release, ROS production, TNF-α, and IL-1β expression, as well as elevated SOD levels. KLF4 was a direct target of miR-212, and miR-212 repressed KLF4 expression in a post-transcriptional manner. Moreover, miR-212-mediated protection effects were abated following KLF4 expression restoration in MPP⁺-induced SH-SY5Y cells, represented as lowered cell viability and enhanced apoptotic rate. Furthermore, Notch signaling was involved in the regulation of miR-212/KLF4 axis in MPP⁺-induced SH-SY5Y cells.
miR-212 might attenuate MPP⁺-induced neuronal damage by regulating KLF4/Notch signaling pathway in SH-SY5Y cells, a promising target for PD therapy.
帕金森病（PD）是一种常见的年龄依赖性神经退行性疾病。已证明miR-212在几种神经疾病中发挥保护作用。本研究旨在探讨miR-212在PD中的作用及潜在分子机制。
将1-甲基-4-苯基吡啶离子（MPP⁺）诱导的SH-SY5Y细胞用作体外PD模型。采用RT-qPCR检测miR-212和克鲁ppel样因子4（KLF4）mRNA的表达。进行蛋白质印迹分析以检测KLF4、Notch1和Jagged1的蛋白水平。分别通过细胞计数试剂盒-8和流式细胞术测定细胞活力和凋亡情况。使用相应的ELISA试剂盒对caspase-3活性、乳酸脱氢酶（LDH）、活性氧（ROS）、超氧化物歧化酶（SOD）、肿瘤坏死因子-α（TNF-α）和白细胞介素-1β（IL-1β）进行定量分析。采用双荧光素酶报告基因检测法评估miR-212与KLF4之间的关系。
在MPP⁺诱导的SH-SY5Y细胞中，miR-212表达下调。此外，miR-212减轻了MPP⁺诱导的SH-SY5Y细胞损伤，表现为细胞活力增加、caspase-3活性降低、LDH释放减少、ROS产生减少、TNF-α和IL-1β表达降低以及SOD水平升高。KLF4是miR-212的直接靶标，miR-212以转录后方式抑制KLF4表达。此外，在MPP⁺诱导的SH-SY5Y细胞中恢复KLF4表达后，miR-212介导的保护作用减弱，表现为细胞活力降低和凋亡率增加。此外，Notch信号通路参与了MPP⁺诱导的SH-SY5Y细胞中miR-212/KLF4轴的调控。
miR-212可能通过调节SH-SY5Y细胞中的KLF4/Notch信号通路减轻MPP⁺诱导的神经元损伤，是PD治疗的一个有前景的靶点。
