Jiang Mingfang, Yun Qiang, Shi Feng, Niu Guangming, Gao Yang, Xie Shenghui, Yu Shengyuan
Department of Neurology, Chinese PLA General Hospital, Beijing, China; Department of Neurology, Affiliated Hospital of Inner Mongolia Medical University, Hohhot, China;
Department of Neurosurgery, Inner Mongolia People's Hospital, Hohhot, China;
Am J Physiol Cell Physiol. 2016 May 1;310(9):C755-63. doi: 10.1152/ajpcell.00226.2015. Epub 2016 Feb 10.
Endoplasmic reticulum (ER) stress has been linked to the pathogenesis of Parkinson's disease (PD). However, the role of microRNAs (miRNAs) in this process involved in PD remains poorly understood. Recent studies indicate that miR-384-5p plays an important role for cell survival in response to different insults, but the role of miR-384-5p in PD-associated neurotoxicity remains unknown. In this study, we investigated the role of miR-384-5p in an in vitro model of PD using dopaminergic SH-SY5Y cells treated with rotenone. We found that miR-384-5p was persistently induced by rotenone in neurons. Also, the inhibition of miR-384-5p significantly suppressed rotenone-induced neurotoxicity, while overexpression of miR-384-5p aggravated rotenone-induced neurotoxicity. Through bioinformatics and dual-luciferase reporter assay, miR-384-5p was found to directly target the 3'-untranslated region of glucose-regulated protein 78 (GRP78), the master regulator of ER stress sensors. Quantitative polymerase chain reaction and Western blotting analysis showed that miR-384-5p negatively regulated the expression of GRP78. Inhibition of miR-384-5p remarkably suppressed rotenone-evoked ER stress, which was evident by a reduction in the phosphorylation of activating transcription factor 4 (ATF4) and inositol-requiring enzyme 1 (IRE1α). The downstream target genes of ER stress including CCAAT/enhancer-binding protein-homologous protein (CHOP) and X box-binding protein-1 (XBP-1) were also decreased by the miR-384-5p inhibitor. In contrast, overexpression of miR-384-5p enhanced ER stress signaling. In addition, knockdown of GRP78 significantly abrogated the inhibitory effect of miR-384-5p inhibitors on cell apoptosis and ER stress signaling. Moreover, we observed a significant increase of miR-384-5p expression in primary neurons induced by rotenone. Taken together, our results suggest that miR-384-5p mediated ER stress by negatively regulating GRP78 and that miR-384-5p inhibition might be a novel and promising approach for the treatment of PD.
内质网（ER）应激与帕金森病（PD）的发病机制有关。然而，微小RNA（miRNA）在PD这一过程中的作用仍知之甚少。最近的研究表明，miR-384-5p在细胞应对不同损伤时的存活中起重要作用，但miR-384-5p在PD相关神经毒性中的作用尚不清楚。在本研究中，我们使用经鱼藤酮处理的多巴胺能SH-SY5Y细胞，在PD体外模型中研究了miR-384-5p的作用。我们发现鱼藤酮在神经元中持续诱导miR-384-5p表达。此外，抑制miR-384-5p可显著抑制鱼藤酮诱导的神经毒性，而miR-384-5p过表达则加重鱼藤酮诱导的神经毒性。通过生物信息学和双荧光素酶报告基因检测，发现miR-384-5p直接靶向内质网应激传感器的主要调节因子葡萄糖调节蛋白78（GRP78）的3'-非翻译区。定量聚合酶链反应和蛋白质印迹分析表明，miR-384-5p负向调节GRP78的表达。抑制miR-384-5p可显著抑制鱼藤酮诱发的内质网应激，这在激活转录因子4（ATF4）和肌醇需求酶1（IRE1α）的磷酸化减少中明显可见。内质网应激的下游靶基因，包括CCAAT/增强子结合蛋白同源蛋白（CHOP）和X盒结合蛋白1（XBP-1），也因miR-384-5p抑制剂而减少。相反，miR-384-5p过表达增强了内质网应激信号。此外，敲低GRP78可显著消除miR-384-5p抑制剂对细胞凋亡和内质网应激信号的抑制作用。此外，我们观察到鱼藤酮诱导的原代神经元中miR-384-5p表达显著增加。综上所述，我们的结果表明，miR-384-5p通过负向调节GRP78介导内质网应激，并且抑制miR-384-5p可能是一种治疗PD的新的有前景的方法。
