Department of Orthopaedics, The First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150001, P.R. China.
Department of Orthopaedics, Binzhou Medical University Hospital, Binzhou, Shandong 256603, P.R. China.
Mol Med Rep. 2021 Sep;24(3). doi: 10.3892/mmr.2021.12306. Epub 2021 Jul 23.
Spinal cord injury (SCI) is characterized by permanent motor deficits followed by inflammation and oxidative stress, causing neuronal cell death. The present study aimed to investigate the role of microRNA (miR)‑128 in neuronal cell apoptosis and its underlying mechanism. Targeting relationships among miR‑128 and Unc‑51 like autophagy activating kinase 1 (ULK1) and Fas ligand (FasL) were verified using dual‑luciferase reporter assay and ChIP assays. Loss‑ and gain‑of‑function assays were conducted in rat models of SCI to determine the roles of miR‑128 and ULK1 in neuronal cell apoptosis, inflammation, and motor function. Apoptosis, motor function and expression of inflammatory factors were respectively determined by Terminal deoxynucleotidyl transferase‑mediated dUTp nick end‑labeling, Basso, Beattie and Bresnahan (BBB) score and enzyme‑linked immunosorbent assay. Hematoxylin and eosin staining, Nissl staining and immunofluorescence were respectively performed to observe morphological changes and number of neurons and nestin‑positive cells. The neuronal cells were isolated from neuron injury models and cultured . MTT and flow cytometry was conducted to determine the neuronal cell viability and apoptosis respectively. miR‑128 was downregulated whereas ULK1 was upregulated in rats with SCI. Overexpression of miR‑128 or downregulation of ULK1 inhibited neuronal cell apoptosis and inflammation as evidenced by an increased BBB score and more neurons and nestin‑positive cells, but reduced expression of inflammatory and apoptosis‑related factors. ULK1 was negatively regulated by miR‑128, whereas FasL was positively regulated by ULK1. experiments validated the roles of miR‑128 and ULK1 in neuronal cell differentiation and apoptosis. In conclusion, the upregulation of miR‑128 depresses neuronal cell apoptosis by downregulating ULK1, thereby attenuating SCI via the downregulation of FasL.
脊髓损伤（SCI）的特征是永久性运动功能障碍，随后发生炎症和氧化应激，导致神经元细胞死亡。本研究旨在探讨微小 RNA（miR）-128 在神经元细胞凋亡中的作用及其潜在机制。通过双荧光素酶报告基因检测和染色质免疫沉淀（ChIP）实验验证了 miR-128 与自噬激活激酶 1（ULK1）和 Fas 配体（FasL）之间的靶向关系。在 SCI 大鼠模型中进行了 miR-128 和 ULK1 的缺失和功能获得实验，以确定 miR-128 和 ULK1 在神经元细胞凋亡、炎症和运动功能中的作用。通过末端脱氧核苷酸转移酶介导的 dUTP 缺口末端标记法、Basso、Beattie 和 Bresnahan（BBB）评分和酶联免疫吸附试验分别测定细胞凋亡、运动功能和炎症因子的表达。苏木精和伊红染色、尼氏染色和免疫荧光分别用于观察形态变化、神经元数量和巢蛋白阳性细胞。从神经元损伤模型中分离神经元细胞并进行培养。通过 MTT 和流式细胞术分别测定神经元细胞活力和凋亡。SCI 大鼠中 miR-128 下调而 ULK1 上调。miR-128 的过表达或 ULK1 的下调抑制神经元细胞凋亡和炎症，表现为 BBB 评分增加、更多的神经元和巢蛋白阳性细胞，以及炎症和凋亡相关因子的表达减少。ULK1 受 miR-128 负调控，而 FasL 受 ULK1 正调控。进一步的实验验证了 miR-128 和 ULK1 在神经元细胞分化和凋亡中的作用。总之，miR-128 的上调通过下调 ULK1 抑制神经元细胞凋亡，从而通过下调 FasL 减轻 SCI。
