Vol 59, No 1 (2021)
Original paper
Published online: 2021-03-02

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

Yun Zhang12, Peilan Zhang1, Chunying Deng13
Pubmed: 33651374
Folia Histochem Cytobiol 2021;59(1):57-65.


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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