Cai-Dong Liu,1,2,* Qiang Peng,3,* Shi-Yao Wang,3,* Yang Deng,2 Zhong-Yuan Li,3 Zhao-Han Xu,3 Liang Wu,3 Ying-Dong Zhang,2,3 Rui Duan3 

1Department of Laboratory Medicine, Nanjing First Hospital, China Pharmaceutical University, Nanjing, Jiangsu, 210006, People’s Republic of China; 2School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu, 210006, People’s Republic of China; 3Department of Neurology, Nanjing First Hospital, Nanjing Medical University, Nanjing, Jiangsu, 210006, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Ying-Dong Zhang; Rui Duan, Department of Neurology, Nanjing First Hospital, Nanjing Medical University, No. 68, Changle Road, Nanjing, Jiangsu, People’s Republic of China, Email zhangyingdong@njmu.edu.cn; duanruicpu@163.com

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