Decoding mitochondrial apoptosis in ischemic stroke via a miR-21a-3p/Plaur-centered regulatory network

Ischemic stroke is a major cause of death and disability, and current therapies only partly limit secondary brain injury driven by apoptosis and oxidative stress. Mitochondria dependent cell death is a key contributor, but the upstream regulatory networks and cell type specific effectors remain unclear.

Single cell RNA sequencing, bulk RNA sequencing and miRNA profiling from mouse middle cerebral artery occlusion models were integrated to identify mitochondrial apoptosis related pathways and key regulators. Cell type composition and differentially expressed genes were analyzed by scRNA-seq, intersected bulk datasets were used to define shared apoptosis related genes, and miRNA target prediction nominated candidate miRNA-mRNA pairs. Hypoxia treated BV2 microglia and bEnd.3 endothelial cells were used for functional validation by qPCR, western blotting, flow cytometry, LDH release, CCK-8 and dual luciferase assays.

scRNA-seq identified twelve brain cell populations and showed that endothelial cells, monocytes, astrocytes and microglia display the most prominent changes after ischemia, with Gene Ontology enrichment repeatedly highlighting "apoptotic mitochondrial changes" (GO:0008637). Intersecting two bulk RNA-seq cohorts recovered the same pathway and four candidates (Hk2, Pycard, Fas and Plaur), with Plaur most strongly dysregulated in endothelial and myeloid cells. miRNA profiling and multi database prediction highlighted miR-21a-3p as a putative upstream regulator of Plaur. In vitro, hypoxia increased Plaur and decreased miR-21a-3p. Plaur silencing reduced apoptosis, reactive oxygen species and LDH release and improved viability, while miR-21a-3p overexpression lowered Plaur and attenuated hypoxia induced injury; inhibition of miR-21a-3p had opposite effects. Dual luciferase assays confirmed direct binding of miR-21a-3p to the Plaur 3'UTR.

The miR-21a-3p/Plaur axis links miRNA regulation to mitochondria associated apoptotic and oxidative damage pathways in ischemia related cellular models and represents a potential target for further mechanistic and translational studies in ischemic stroke.