open access

Vol 59, No 1 (2021)
Original paper
Submitted: 2020-07-08
Accepted: 2021-02-22
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
DOI: 10.5603/FHC.a2021.0007
·
Pubmed: 33651374
·
Folia Histochem Cytobiol 2021;59(1):57-65.
Affiliations
  1. Clinical College of Neurology, Neurosurgery and Neurorehabilitation, Tianjin Medical University, 300350, China
  2. Department of Geriatrics, Tangshan Gongren Hospital, Tangshan City, Hebei Province, 063000, China
  3. Department of Neurology, North China University of Science and Technology Affiliated Hospital, Tangshan City, Hebei Province, 063000, China

open access

Vol 59, No 1 (2021)
ORIGINAL PAPERS
Submitted: 2020-07-08
Accepted: 2021-02-22
Published online: 2021-03-02

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.

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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Keywords

miR-378a-5p; primary hippocampal neurons; rat; oxygen-glucose deprivation/reoxygenation; CAMKK2/AMPK pathway; apoptosis

About this article
Title

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

Journal

Folia Histochemica et Cytobiologica

Issue

Vol 59, No 1 (2021)

Article type

Original paper

Pages

57-65

Published online

2021-03-02

DOI

10.5603/FHC.a2021.0007

Pubmed

33651374

Bibliographic record

Folia Histochem Cytobiol 2021;59(1):57-65.

Keywords

miR-378a-5p
primary hippocampal neurons
rat
oxygen-glucose deprivation/reoxygenation
CAMKK2/AMPK pathway
apoptosis

Authors

Yun Zhang
Peilan Zhang
Chunying Deng

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