Circ_0008146 Exacerbates Ferroptosis via Regulating the miR-342-5p/ACSL4 Axis After Cerebral Ischemic/Reperfusion

Abstract

Purpose

Acute ischemic stroke (AIS) has seriously threatened people’s health worldwide and there is an urge need for early diagnosis and effective treatment of AIS. This research intended to clarify the regulatory role of circ_0008146/miR-342-5p/ACSL4 axis in AIS.

Methods

High-throughput small RNA sequencing analysis was adapted to identify differentially expressed miRNAs between the AIS and control group. The circ_0008146, miR-342-5p, and ACSL4 levels were detected by qRT-PCR. Middle cerebral artery occlusion/reperfusion (MCAO/R) models were constructed in C57BL/6J mice. Assay kits were used to determine Fe²⁺ levels and a battery of oxidative stress and lipid peroxidation indicators, including ROS, MDA, LPO, SOD and GSH/GSSG ratio. The protein levels of ACSL4 were measured by Western blot. The behavioral function was assessed using neurobehavioral tests. TTC staining was employed to visualize infarction size. Nissl staining was adapted to detect histopathological changes. Receiver operating characteristic curve and correlation analysis were applied to investigate the clinical value and association of miR-342-5p and ACSL4.

Results

A total of 44 AIS patients and 49 healthy controls were enrolled in our study. The small RNA sequencing unveiled a significant decrease in miR-342-5p levels in AIS patients. MiR-342-5p inhibited oxidative stress and RSL3-induced ferroptosis after cerebral ischemic/reperfusion injury in vivo by targeting ferroptosis-related gene ACSL4. Circ_0008146 acted as a sponge of miR-342-5p, and overexpression of circ_0008146 increased neurological deficits and brain injury in mice. Circ_0008146 contributed to ferroptosis in cerebral infarction via sponging miR-342-5p to regulate ACSL4. Plasma miR-342-5p and ACSL4 demonstrated significant correlation and good diagnostic value for AIS patients.

Conclusion

This study provides the first in vivo evidence to show that circ_0008146 exacerbates neuronal ferroptosis after AIS via the miR-342-5p/ACSL4 axis. Furthermore, miR-342-5p/ACSL4 axis holds promise as a viable therapeutic target and practical biomarkers for AIS patients.

Keywords:

• ischemic stroke
• miRNA
• circRNA
• ferroptosis
• biomarker

Data Sharing Statement

The data that support this study are available from the corresponding author Rui Duan upon reasonable request.

Ethical Approval

This study was in full compliance with the Declaration of Helsinki and was approved by the Ethics Committee of Nanjing First Hospital, Nanjing Medical University (No. KY20211011-05). All study participants provided written informed consent before enrollment in the study. This study was also approved by the Laboratory Animal Ethics Committee of Nanjing First Hospital, Nanjing Medical University (No. DWSY-23146450). The experimental protocol was conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors report no conflicts of interest in this work.

Additional information

Funding

This study was funded by Jiangsu Provincial Medical Key Discipline Cultivation Unit (JSDW202239) and Key Project supported by Medical Science and technology development Foundation, Nanjing Department of Health (YKK22107).