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Circular RNA 0025984 Ameliorates Ischemic Stroke Injury and Protects Astrocytes Through miR-143-3p/TET1/ORP150 Pathway

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Abstract

MiR-143-3p is aberrantly expressed in patients with ischemic stroke and associated with ischemic brain injury. However, the underlying mechanisms are largely unknown. Here, we confirmed circ_0025984 and TET1 as a sponge and target of miR-143-3p, respectively, by luciferase reporter assay. In astrocytes, OGD significantly decreased circ_0025984 and TET1 levels but increased miR-143-3p levels, which was also observed in brains of mice with MCAO. Treatment with miR-143-3p inhibitor or circ_0025984 significantly decreased astrocyte apoptosis and autophagy, as well as cerebral injury and neuron loss in mice with MCAO. Notably, TET1 overexpression decreased astrocyte apoptosis and autophagy and induced promoter hypomethylation and expression of ORP150. Our results demonstrated for the first time that circ_0025984 protects astrocytes from ischemia-induced autophagy and apoptosis by targeting the miR-143-3p/TET1 pathway and might inhibit cerebral injury induced by ischemic stroke. Furthermore, our data revealed the important positive regulation of ORP150 by TET1, which could be associated with its neuroprotective role.

Model for signaling pathway of circ_0025984/miR-143-3p/TET1 inastrocytes cultured under OGD. In astrocytes, circ_0025984 acts as a sponge of miR-143-3p, which directly targets TET1 and decreases its expression (A). After translocatinginto the nucleus, TET1 binds to the promoter of ORP150, converts 5mC into 5hmC,leading to DNA demethylation and increased expression of ORP150 (B). In astrocytescultured under OGD, ER stress is induced and eventually leads to apoptosis andautophagy mediated by ATG7, which is regulated by circ_0025984 via ORP150 andGRP78 (C).

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Data availability

All data generated or analyzed during this study are available from the corresponding author upon reasonable request.

Abbreviations

rt-PA:

Recombinant tissue plasminogen activator

CNS:

Central nervous system

circRNAs:

Circular RNAs

tMCAO:

Transient middle cerebral artery occlusion

MCAO:

Middle cerebral artery occlusion

CCA:

Common carotid artery

PFA:

Paraformaldehyde

DMEM:

Dulbecco’s modified Eagle medium

TET1:

Ten-eleven translocation methylcytosine dioxygenase 1

NCs:

Negative controls

OGD:

Oxygen and glucose deprivation

TEM:

Transmission electron microscope

FISH:

Fluorescence in situ hybridization

ChIP:

Chromatin immunoprecipitation

RIP:

RNA immunoprecipitation

MSP:

Methylation-specific PCR

5mC:

5-Methyl-cytosine

5hmC:

5-Hydroxymethyl-cytosine

FGF:

Fibroblast growth factor

BDNF:

Brain-derived neurotrophic factor

ER:

Endoplasmic reticulum

HMEC-1:

Human microvascular endothelial cells-1

oxLDL:

Oxidized low density lipoprotein

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Funding

This study was supported by the National Natural Science Foundation of China (No. 81860321).

The follow-up supplementary experiment for this work is supported by Regional common diseases and adult stem cell transformation research innovation platform, Science and Technology Department of Guizhou Province [Guizhou specific grant (2019) 4008].

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Author notes

  1. Daixuan Zhou and Zhi Huang contributed equally to this work.

Authors and Affiliations

  1. Queen Mary College, Nanchang University, Nanchang, 330031, People’s Republic of China

    Daixuan Zhou

  2. School of Basic Medical Science, Guizhou Medical University, Guiyang, 550002, People’s Republic of China

    Zhi Huang

  3. Key Laboratory of Endemic and Ethnic Diseases, Ministry of Education, Guizhou Medical University, Guiyang, 550002, People’s Republic of China

    Xiaoxi Zhu

  4. Department of Neurosurgery, The First Affiliated Hospital of Nanchang University, Nanchang, 330029, People’s Republic of China

    Tao Hong

  5. Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Nanchang, 100070, People’s Republic of China

    Yuanli Zhao

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  1. Daixuan Zhou
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Contributions

Daixuan Zhou, Zhi Huang, and Xiaoxi Zhu designed the experiments; Daixuan Zhou and Zhi Huang performed the experiments. All authors analyzed the data and prepared the manuscript.

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Correspondence to Tao Hong or Yuanli Zhao.

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This study was approved by the Medical Ethics Committee of the Affiliated Hospital of Guizhou Medical University.

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Supplementary Information

Supplementary Figure 1.

Circ_0025984 and TET1 expression in the hippocampus of the brain. Circ_0025984 level was confirmed through FISH assay in the hippocampus of the brain (A). TET1 expression was determined by IF assay in the hippocampus of the brain (B). (PNG 3349 kb)

High resolution image (TIF 3257 kb)

Supplementary Figure 2.

MiR-143-3p inhibition dramatically induced neuron growth and alleviated cerebral injury in MCAO model rats. After miR-143-3p inhibition, western blot assay was adopted to evaluate c-Myc and Cyclin D1 expressions in MCAO model rats (A). And we also quantitatively analyzed the relative expressions of c-Myc and Cyclin D1 in line with the grayscale (B). After treatment with miR-143-3p inhibitor, the infarct volume was calculated through TTC staining (C). The mNSS scores were also assessed at 1, 3, 7 and 14 days in MCAO model rats (D). *p < 0.05, **p < 0.01, ***p < 0.001. (PNG 679 kb)

High resolution image (TIF 8726 kb)

Supplementary Figure 3.

Circ_0025984 overexpression prominently promoted neuron growth and relieved cerebral injury in MCAO model rats. c-Myc and Cyclin D1 expressions were examined by western blot assay in MCAO model rats after circ_0025984 overexpression (A). The relative levels of c-Myc and Cyclin D1 were calculated based on the grayscale (B). We also evaluated the infarct volume (C) and mNSS scores (D) in MCAO model rats after circ_0025984 overexpression. *p < 0.05, **p < 0.01, ***p < 0.001. (PNG 682 kb)

High resolution image (TIF 8726 kb)

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Zhou, D., Huang, Z., Zhu, X. et al. Circular RNA 0025984 Ameliorates Ischemic Stroke Injury and Protects Astrocytes Through miR-143-3p/TET1/ORP150 Pathway. Mol Neurobiol 58, 5937–5953 (2021). https://doi.org/10.1007/s12035-021-02486-8

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