MiR-4472 serves as a potential biomarker for hypoxic-ischemic encephalopathy and promotes neuronal death as well as hypoxic-ischemic brain damage in neonatal rats by targeting MEF2D

Background: Hypoxic-ischemic encephalopathy (HIE) is a predominant cause in neonatal mortality and long-term neurological impairment. Accumulating evidence underscores the pivotal involvement of MicroRNAs (miRNAs) in HIE. Our prior miRNA microarray analysis revealed elevated miR-4472 levels in plasma from HIE newborns. However, the diagnostic potential of miR-4472 for HIE and its mechanistic contributions to disease progression remain to be elucidated.

Methods: We collected plasma samples from HIE and healthy newborns, qRT-PCR and receiver operating characteristic (ROC) curve analyses were employed to evaluate miR-4472 expression and diagnostic performance, respectively. An in vitro HIE model was established using oxygen-glucose deprivation and reperfusion (OGD/R)-treated SH-SY5Y cells, while an in vivo HIE model was generated by subjecting seven days old male SD rats to carotid artery ligation followed by hypoxia. Neurological function in HIE neonatal rats was assessed using the Zea-Longa scoring system and Morris Water Maze tests, whereas brain tissue pathology and Apoptosis were analyzed through HE staining and TUNEL assays, respectively. The targeting relationship between miR-4472 and MEF2D was validated via western blotting and dual-luciferase reporter assays. CCK-8 and Annexin V-FITC/PI staining were utilized to elucidate the effects of miR-4472 on SH-SY5Y cell viability and Apoptosis through MEF2D. ELISA and commercial kits were used to quantify inflammatory factors, ROS, and MDA levels.

Results: Our study demonstrated that miR-4472 was significantly elevated in plasma from HIE newborns, OGD/R-treated SH-SY5Y cells, and brain tissue of HIE rats, with higher levels observed in moderate-to-severe cases compared to mild HIE, the area under curve (AUC) of miR-4472 for HIE diagnosis reaching 0.958. Inhibition of miR-4472 restored cell viability and reduced Apoptosis in OGD/R-treated SH-SY5Y cells, improved neurological function and ameliorated brain damage in HIE rats. Mechanistically, miR-4472 directly targets and suppresses MEF2D expression, thereby increasing ROS and MDA levels, inducing the expression and release of IL-6, TNF-α, and IL-1β. Intervention with MEF2D effectively antagonized the effects of miR-4472 in HIE.

Conclusion: This study demonstrates that plasma miR-4472 serves as a potential diagnostic biomarker for HIE and promotes its pathological progression by targeting MEF2D.

HIE; Inflammation; MEF2D; Oxidative stress; miR-4472.