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Blockage of miR-485-5p on Cortical Neuronal Apoptosis Induced by Oxygen and Glucose Deprivation/Reoxygenation Through Inactivating MAPK Pathway

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Abstract

This study is designed to explore the role of miR-485-5p in hypoxia/reoxygenation-induced neuronal injury in primary rat cortical neurons. Hypoxia/reoxygenation model was established through oxygen and glucose deprivation/reoxygenation (OGD/R). RN-c cells were transfected with miR-485-5p mimics, miR-485-5p inhibitors, si-SOX6, pCNDA3.1-SOX6 or miR-485-5p + pCDNA3.1-SOX6, in which cell viability, apoptosis, lactate dehydrogenase (LDH) release rate were assessed. Western blot detected the protein expressions of apoptotic-related proteins (caspase3, Bcl-2, Bax) and the phosphorylated level of ERK1/2. The potential binding sites between miR-485-5p and SOX6 were predicted by STARBASE and identified using dual luciferase reporter gene assay. OGD/R-treated RN-c cell presented increases in apoptosis and LDH release rate as well as a decrease in cell viability. miR-485-5p was downregulated while SOX6 was upregulated in OGD/R-treated RN-c cells. Overexpression of miR-485-5p or SOX6 knockdown rescued cell viability and Bcl-2 expression, while attenuated apoptosis, LDH release rate, expression of SOX6 and the phosphorylated level of ERK1/2. Consistently, miR-485-5p inhibition led to the reverse pattern. Co-transfection of miR-485-5p and SOX6 reversed the protective effect of miR-485-5p on OGD/R-induced neuronal apoptosis. miR-485-5p can directly target SOX6. Together, miR-485-5p inhibited SOX6 to alleviate OGD/R-induced apoptosis. Collectively, miR-485-5p protects primary cortical neurons against hypoxia injury through downregulating SOX6 and inhibiting MAPK pathway.

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Abbreviations

OGD/R:

Oxygen and glucose deprivation/reoxygenation

LDH:

Lactate dehydrogenase

miRNAs:

MicroRNAs

FBS:

Fetal bovine serum

PBS:

Phosphate buffer saline

DMEM:

Dulbecco’s modified eagle medium

MTT:

Methyl thiazolyl tetrazolium

OD:

Optical density

EDU:

5-Ethynyl-2′-deoxyuridine

FCM:

Flow cytometry

PI:

Prodium iodide

qRT-PCR:

Quantitative real time polymerase chain reaction

BCA:

Bicinchoninic acid

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

PVDF:

Polyvinylidene fluoride

ECL:

Enhanced chemiluminescence

CNS:

Central nerve system

3′-UTR:

3′Untranslated regions

NMDA:

N-methyl-d-aspartate

SVHRP:

Scorpion venom heat-resistant peptide

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Acknowledgements

This work was supported by the Grant from Hunan Provincial Natural Science Foundation (2018JJ6086).

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Authors and Affiliations

  1. Department of Neurosurgery, Changsha Central Hospital of University of South China, Changsha, 410005, Hunan, People’s Republic of China

    Jiangliu Yin, Suonan Li & Shuai Zhang

  2. Hunan Provincial Center for Disease Prevent and Control, Changsha, 410006, Hunan, People’s Republic of China

    Huan Chen

  3. Department of Neurosurgery, Jinjiang Municipal Hospital of Quanzhou Medical College, No. 392, Xinhua Road, Meiling Street, Quanzhou, 362200, Fujian, People’s Republic of China

    Xieli Guo

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  1. Jiangliu Yin
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  3. Suonan Li
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  4. Shuai Zhang
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Contributions

YJL and GXL conceived the ideas. YJL designed the experiments. CH performed the experiments. LSN analyzed the data. GXL and ZS provided critical materials. YJL and CH wrote the manuscript. GXL supervised the study. All the authors have read and approved the final version for publication.

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Correspondence to Xieli Guo.

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Yin, J., Chen, H., Li, S. et al. Blockage of miR-485-5p on Cortical Neuronal Apoptosis Induced by Oxygen and Glucose Deprivation/Reoxygenation Through Inactivating MAPK Pathway. Neuromol Med 23, 256–266 (2021). https://doi.org/10.1007/s12017-020-08605-3

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  • DOI: https://doi.org/10.1007/s12017-020-08605-3

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