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MiR-19b-3p Inhibits Hypoxia-Ischemia Encephalopathy by Inhibiting SOX6 Expression via Activating Wnt/β-catenin Pathway

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Abstract

Hypoxic-ischemic encephalopathy (HIE) is a detrimental factor in infant death and chronic disease. The specific pathogenesis is not entirely clear. Therefore, exploring the pathogenesis of HIE is critical. The expression of miR-19b-3p and SOX6 in umbilical blood of HIE patients was detected by qRT-PCR assay. HT22 cells were triggered with oxygen-glucose deprivation/reoxygenation (OGD/R) to construct the HIE cell model. Cell Counting Kit-8 (CCK-8) assay was used to estimate viability. SOD and MDA levels were detected by enzyme linked immunosorbent assay. Flow cytometry was implemented to ascertain neurocyte apoptosis. Cellular β-catenin immunofluorescence staining was used to detect the expression and distribution of β-catenin protein. Wnt signaling pathway activation was detected by TOPFlash/FOPFlash luciferase reporter assay. The targeting correlation of SOX6 and miR-19b-3p was corroborated by dual-luciferase reporter gene assay and RNA pull-down assay. MiR-19b-3p expression was once down-regulated, whilst SOX6 expression was up-regulated in HIE patients. MiR-19b-3p overexpression promoted cell proliferation, repressed cell apoptosis, oxidative stress response, and Wnt/β-catenin pathway activation in OGD/R-triggered HT22 cells. MiR-19b-3p negatively regulated SOX6 expression. SOX6 knockdown improved OGD/R-triggered HT22 cells injury via Wnt/β-catenin pathway activation. MiR-19b-3p overexpression suppressed OGD/R-triggered HT22 cell injury via inhibiting SOX6 expression via activating Wnt/β-catenin pathway.

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Abbreviations

DEME:

Dulbecco’s modified eagle medium

FBS:

Fetal bovine serum

HIBD:

Hypoxic-ischemic brain damage

HIE:

Hypoxic-ischemic encephalopathy

miRNAs:

MicroRNAs

miR-19b-3p:

MicroRNA-19b-3p

PBS:

Phosphate buffered saline

PVDF:

Poly vinylidene difluoride flter

qRT-PCR:

Quantitative real-time PCR

SDS-PGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SOX6:

Sex-determining region Y box6

WB:

Western blot analysis

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Acknowledgements

We would like to give our sincere gratitude to the reviewers for their constructive comments.

Funding

This work was supported by District-level scientific research project of medical and health institutions in Longhua district of Shenzhen (Project No. 2020191), and Basic Research Project of Shenzhen Science and Technology Innovation Committee (Project No. JCYJ20190808154015736).

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Authors and Affiliations

  1. Department of Neurosurgery, Shenzhen Longhua District Central Hospital, Shenzhen, 518110, Guangdong, People’s Republic of China

    Hao Zeng

  2. Department of Neontal Development, Shenzhen Longhua District Central Hospital, No. 187, Guanlan Avenue, Longhua District, Shenzhen, 518110, Guangdong, People’s Republic of China

    Yu-Xia Chen

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Correspondence to Yu-Xia Chen.

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Supplementary Information

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11064_2022_3812_MOESM1_ESM.tif

Supplementary material 1 (TIF 1974 kb) Figure S1. OGD/R-triggered HT22 cells were transfected with miR-19b-3p mimics and/or Oe-SOX6. (A) Luciferase reporter gene assay was used to confirm the targeting relationship of miR-19b-3p with FOXP2, EBF2, SOX6, PLXNC1, BTBD7. (B) IF staining was used to ascertain cellular β-catenin expression and distribution in corresponding group. (C) Wnt signaling pathway activation was ascertained by TOPFlash/FOPFlash luciferase reporter assay. At least three independent behavior experiments were performed in each group. **P < 0.01, compared to labelled groups

11064_2022_3812_MOESM2_ESM.tif

Supplementary material 2 (TIF 1817 kb) Figure S2. OGD/R-triggered HT22 cells were transfected with Oe-SOX6. (A and B) SOX6 mRNA and protein expression was estimated by qRT-PCR assay and WB. (C) Cell viability was detected by CCK-8 assay. (D) SOD and MDA levels were ascertained by ELISA assay. (E and F) Apoptosis rate and the expression levels of related protein were ascertained by flow cytometry and WB. At least three independent behavior experiments were performed in each group. **P < 0.01, compared to labelled groups

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Zeng, H., Chen, YX. MiR-19b-3p Inhibits Hypoxia-Ischemia Encephalopathy by Inhibiting SOX6 Expression via Activating Wnt/β-catenin Pathway. Neurochem Res 48, 874–884 (2023). https://doi.org/10.1007/s11064-022-03812-9

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