Yonsei Med J.  2022 Apr;63(4):389-398. 10.3349/ymj.2022.63.4.389.

MiR-494-3p Upregulation Exacerbates Cerebral Ischemia Injury by Targeting Bhlhe40

• Sun L
• Ji D
• Zhi F
• Fang Y
• Zhu Z
• Ni T
• Zhu Q
• Bao J

• KMID: 2527521
• DOI: http://doi.org/10.3349/ymj.2022.63.4.389

Abstract

Purpose
Cerebral ischemia is related to insufficient blood supply and is characterized by abnormal reactive oxygen species (ROS) production and cell apoptosis. Previous studies have revealed a key role for basic helix-loop-helix family member e40 (Bhlhe40) in oxidative stress and cell apoptosis. This study aimed to investigate the roles of miR-494-3p in cerebral ischemia/reperfusion (I/R) injury.
Materials and Methods
A mouse middle cerebral artery occlusion (MCAO/R) model was established to mimic cerebral ischemia in vivo. Brain infarct area was assessed using triphenyl tetrazolium chloride staining. Oxygen-glucose deprivation/reoxygenation (OGD/R) operation was adopted to mimic neuronal injury in vitro. Cell apoptosis was analyzed by flow cytometry. The relationship between miR-494-3p and Bhlhe40 was validated by luciferase reporter and RNA immunoprecipitation assays.
Results
Bhlhe40 expression was downregulated both in MCAO/R animal models and OGD/R-induced SH-SY5Y cells. Bhlhe40 overexpression inhibited cell apoptosis and reduced ROS production in SH-SY5Y cells after OGD/R treatment. MiR-494-3p was verified to bind to Bhlhe40 and negatively regulate Bhlhe40 expression. Additionally, cell apoptosis and ROS production in OGD/ R-treated SH-SY5Y cells were accelerated by miR-494-3p overexpression. Rescue experiments suggested that Bhlhe40 could reverse the effects of miR-494-3p overexpression on ROS production and cell apoptosis.
Conclusion
MiR-494-3p exacerbates brain injury and neuronal injury by regulating Bhlhe40 after I/R.

Keyword

Cerebral I/R injury; Bhlhe40; miR-494-3p; ROS production