Circular RNA HIPK2 Promotes A1 Astrocyte Activation after Spinal Cord Injury through Autophagy and Endoplasmic Reticulum Stress by Modulating miR-124-3p-Mediated Smad2 Repression.

ABSTRACT

Glial scarring formed by reactive astrocytes after spinal cord injury (SCI) is the primary obstacle to neuronal regeneration within the central nervous system, making them a promising target for SCI treatment. Our previous studies have demonstrated the positive impact of miR-124-3p on neuronal repair, but it remains unclear how miR-124-3p is involved in autophagy or ER stress in astrocyte activation. To answer this question, the expression of A1 astrocyte-related markers at the transcriptional and protein levels after SCI was checked in RNA-sequencing data and verified using quantitative polymerase chain reaction (qPCR) and Western blotting in vitro and in vivo. The potential interactions among circHIPK2, miR-124-3p, and Smad2 were analyzed and confirmed by bioinformatics analyses and a luciferase reporter assay. In the end, the role of miR-124-3p in autophagy, ER stress, and SCI was investigated by using Western blotting to measure key biomarkers (C3, LC3, and Chop) in the absence or presence of corresponding selective inhibitors (siRNA, 4-PBA, TG). As a result, SCI caused the increase of A1 astrocyte markers, in which the upregulated circHIPK2 directly targeted miR-124-3p, and the direct downregulating effect of Smad2 by miR-124-3p was abolished, while Agomir-124 treatment reversed this effect. Injury caused a significant change of markers for ER stress and autophagy through the circHIPK2/miR-124-3p/Smad2 pathway, which might activate the A1 phenotype, and ER stress might promote autophagy in astrocytes. In conclusion, circHIPK2 may play a functional role in sequestering miR-124-3p and facilitating the activation of A1 astrocytes through regulating Smad2-mediated downstream autophagy and ER stress pathways, providing a new perspective on potential targets for functional recovery after SCI.