Clinical College of Neurology, Neurosurgery and Neurorehabilitation, Tianjin Medical University, 300350, China.
Department of Geriatrics, Tangshan Gongren Hospital, Tangshan City, Hebei Province, 063000, China.
Folia Histochem Cytobiol. 2021;59(1):57-65. doi: 10.5603/FHC.a2021.0007. Epub 2021 Mar 2.
The pathological mechanism of cerebral ischemia/reperfusion (CIR) injury is complicated and unclear. Apart from the involvement of many low-molecular factors it was found that several miRNAs were dysregulated during and after CIR injury in cell models. This study aimed to explore the effects of miR-378a-5p on in vitro model of (CIR) injury-induced neuronal apoptosis and provide a new mechanism of CIR injury.
Primary hippocampal neurons were isolated from newborn Sprague-Dawley rats. Oxygen- glucose deprivation/reoxygenation (OGDR) for 24 h and 48 h was used as an in vitro model of CIR. Cell viability was measured using MTT assay and apoptosis was determined by flow cytometry. Quantitative real time PCR (qRT-PCR) assay and Western blotting were used to examine mRNA and protein expressions, respectively. TargetScan was used to predict the direct target of miR-378a-5p and luciferase assay was used to validate that calmodulin-dependent protein kinase kinase-2 (CAMKK2) was the direct target of miR-378a-5p.
miR-378a-5p expression was significantly increased after OGDR at 24 h and 48 h. After OGDR, cell viability was reduced, which was reversed by miR-378a-5p and enhanced by shCAMKK2 plasmid. Cell apoptosis was increased after OGDR, which was prevented by miR-378a-5p and enhanced by shCAMKK2 plasmid. Results of TargetScan and luciferase assay demonstrated that miR-378a-5p could directly bind to 3'-untranslated region (3'-UTR) of CAMKK2. Both mRNA and protein expression of CAMKK2 were downregulated by miR-378a-5p mimics and upregulated by miR-378a-5p inhibitors. Phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) was positively associated with expression of CAMKK2.
Data of this study indicated that miR-378a-5p was significantly overexpressed after OGDR. miR-378a-5p could bind to 3'-UTR of CAMKK2 to inhibit cell proliferation through regulation of CAMKK2/AMPK pathway providing a new mechanism and biomarker for the diagnosis and potential treatment of CIR injury.
脑缺血再灌注（CIR）损伤的病理机制复杂且尚未阐明。除了涉及许多低分子因子外，在细胞模型中还发现几种 miRNA 在 CIR 损伤期间和之后失调。本研究旨在探讨 miR-378a-5p 对体外 CIR 损伤诱导的神经元凋亡模型的影响，并为 CIR 损伤提供新的机制。
原代海马神经元从新生 Sprague-Dawley 大鼠中分离出来。用氧葡萄糖剥夺/复氧（OGDR）24 小时和 48 小时作为体外 CIR 模型。用 MTT 法测定细胞活力，用流式细胞术测定细胞凋亡。用定量实时 PCR（qRT-PCR）检测 mRNA 表达，用 Western blot 检测蛋白表达。TargetScan 用于预测 miR-378a-5p 的直接靶标，并用荧光素酶测定验证钙调蛋白依赖性蛋白激酶激酶-2（CAMKK2）是 miR-378a-5p 的直接靶标。
OGDR 24 小时和 48 小时后，miR-378a-5p 的表达明显增加。OGDR 后，细胞活力降低，miR-378a-5p 逆转，shCAMKK2 质粒增强。OGDR 后细胞凋亡增加，miR-378a-5p 可预防，shCAMKK2 质粒增强。TargetScan 和荧光素酶检测结果表明，miR-378a-5p 可直接与 CAMKK2 的 3'-非翻译区（3'-UTR）结合。miR-378a-5p 模拟物下调 CAMKK2 的 mRNA 和蛋白表达，miR-378a-5p 抑制剂上调 CAMKK2 的表达。腺苷单磷酸激活蛋白激酶（AMPK）的磷酸化与 CAMKK2 的表达呈正相关。
本研究数据表明，OGDR 后 miR-378a-5p 表达明显上调。miR-378a-5p 可与 CAMKK2 的 3'-UTR 结合，通过调节 CAMKK2/AMPK 通路抑制细胞增殖，为 CIR 损伤的诊断和潜在治疗提供新的机制和生物标志物。
