Department of Neurology, The First Affiliated Hospital of Zhengzhou University, No. 1, East Jianshe Rd, Zhengzhou, 450052, Henan, China.
Department of Anesthesiology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China.
Neurochem Res. 2021 May;46(5):1119-1128. doi: 10.1007/s11064-021-03245-w. Epub 2021 Feb 9.
This study aimed to explore the effects and function of microRNA-101a-3p (miR-101a-3p) in epilepsy. Rat model of pilocarpine-induced epilepsy was established and the seizure frequency was recorded. Expression of miR-101a-3p and c-Fos in hippocampus tissues of Rat models were detected by qRT-PCR and western blot. Besides, we established a hippocampal neuronal culture model of acquired epilepsy using Mg free medium to evaluate the effects of miR-101a-3p and c-Fos in vitro. Cells were transfected with miR-101a-3p mimic, si-c-FOS, miR-101a-3p + c-FOS and its corresponding controls. MTT assay was used to detect cell viability upon transfection. Flow cytometry was performed to determine the apoptosis rate. Western blot was performed to measure the protein expression of apoptosis-related proteins (Bcl-2, Bax, and cleaved caspase 3), autophagy-related proteins (LC3 and Beclin1) and c-FOS. The targeting relationship between miR-101a-3p and c-FOS was predicted and verified by TargetScan software and dual-luciferase reporter assay. The role of miR-101a-3p was validated using epilepsy rat models in vivo. Another Rat models of pilocarpine-induced epilepsy with miR-NC or miR-101a-3p injection were established to evaluate the effect of miR-101a-3p overexpression on epilepsy in vivo. MiR-101a-3p was downregulated while c-FOS was increased in hippocampus tissues of Rat model of pilocarpine-induced epilepsy. Overexpression of miR-101a-3p or c-FOS depletion promoted cell viability, inhibited cell apoptosis and autophagy. C-FOS was a target of miR-101a-3p and miR-101a-3p negatively regulated c-FOS expression to function in epilepsy. Overexpression of miR-101a-3p attenuated pilocarpine-induced epilepsy in Rats in vivo. This study indicated that miR-101a-3p could attenuate pilocarpine-induced epilepsy by repressing c-Fos expression.
本研究旨在探讨微小 RNA-101a-3p（miR-101a-3p）在癫痫中的作用和功能。建立匹鲁卡品诱导的癫痫大鼠模型，记录癫痫发作频率。通过 qRT-PCR 和 Western blot 检测大鼠模型海马组织中 miR-101a-3p 和 c-Fos 的表达。此外，我们使用无镁培养基建立了获得性癫痫的海马神经元培养模型，以评估 miR-101a-3p 和 c-Fos 在体外的作用。将 miR-101a-3p 模拟物、si-c-FOS、miR-101a-3p+c-FOS 及其相应对照转染细胞。MTT 法检测转染后细胞活力。流式细胞术检测细胞凋亡率。Western blot 法检测凋亡相关蛋白（Bcl-2、Bax 和 cleaved caspase 3）、自噬相关蛋白（LC3 和 Beclin1）和 c-FOS 的蛋白表达。通过 TargetScan 软件和双荧光素酶报告基因检测预测和验证 miR-101a-3p 与 c-FOS 的靶向关系。通过体内癫痫大鼠模型验证 miR-101a-3p 的作用。建立另一种携带 miR-NC 或 miR-101a-3p 注射的匹鲁卡品诱导的癫痫大鼠模型，以评估 miR-101a-3p 过表达对体内癫痫的影响。在匹鲁卡品诱导的癫痫大鼠模型的海马组织中，miR-101a-3p 下调，c-FOS 增加。miR-101a-3p 的过表达或 c-FOS 的耗竭促进了细胞活力，抑制了细胞凋亡和自噬。c-FOS 是 miR-101a-3p 的靶标，miR-101a-3p 负调控 c-FOS 的表达，从而在癫痫中发挥作用。miR-101a-3p 的过表达减轻了体内匹鲁卡品诱导的癫痫发作。本研究表明，miR-101a-3p 通过抑制 c-Fos 表达来减轻匹鲁卡品诱导的癫痫。
