miR-378a-5p regulates CAMKK2/AMPK pathway to contribute to cerebral ischemia/reperfusion injury-induced neuronal apoptosis.

IF 1.7 4区生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY

Folia histochemica et cytobiologica Pub Date : 2021-01-01 Epub Date: 2021-03-02 DOI:10.5603/FHC.a2021.0007

Yun Zhang, Peilan Zhang, Chunying Deng

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引用次数: 0

Abstract

Introduction: 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.

Material and methods: 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.

Results: 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.

Conclusions: 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.

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miR-378a-5p调节CAMKK2/AMPK通路，促进脑缺血再灌注损伤诱导的神经细胞凋亡。

简介脑缺血再灌注（CIR）损伤的病理机制复杂而不清楚。除了许多低分子因素的参与外，研究还发现在细胞模型中，一些 miRNA 在 CIR 损伤过程中和损伤后出现失调。本研究旨在探讨 miR-378a-5p 对体外模型（CIR）损伤诱导的神经细胞凋亡的影响，并提供 CIR 损伤的新机制：从新生Sprague-Dawley大鼠中分离原代海马神经元。材料和方法：从新生 Sprague-Dawley 大鼠体内分离出原代海马神经元，采用氧-葡萄糖剥夺/复氧（OGDR）24 小时和 48 小时作为 CIR 的体外模型。细胞活力用 MTT 法测定，细胞凋亡用流式细胞术测定。定量实时 PCR（qRT-PCR）分析和 Western 印迹分别用于检测 mRNA 和蛋白质的表达。结果：miR-378a-5p表达在OGDR后24 h和48 h显著增加。OGDR 后细胞凋亡增加，miR-378a-5p 阻止了细胞凋亡，而 shCAMKK2 质粒则增强了细胞凋亡。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模拟物可下调CAMKK2的mRNA和蛋白表达，而miR-378a-5p抑制剂可上调CAMKK2的mRNA和蛋白表达。单磷酸腺苷激活蛋白激酶（AMPK）的磷酸化与 CAMKK2 的表达呈正相关：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了新的机制和生物标志物。

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来源期刊

Folia histochemica et cytobiologica 生物-生化与分子生物学

CiteScore

2.80

自引率

6.70%

发文量

审稿时长

6-12 weeks

期刊介绍： "Folia Histochemica et Cytobiologica" is an international, English-language journal publishing articles in the areas of histochemistry, cytochemistry and cell & tissue biology. "Folia Histochemica et Cytobiologica" was established in 1963 under the title: ‘Folia Histochemica et Cytochemica’ by the Polish Histochemical and Cytochemical Society as a journal devoted to the rapidly developing fields of histochemistry and cytochemistry. In 1984, the profile of the journal was broadened to accommodate papers dealing with cell and tissue biology, and the title was accordingly changed to "Folia Histochemica et Cytobiologica". "Folia Histochemica et Cytobiologica" is published quarterly, one volume a year, by the Polish Histochemical and Cytochemical Society.