Stress-Associated Changes in MiR-20b-3p as a Potential Predictor of Underlying Psychopathology in the Depressed Brain: Mechanistic Insights from a Rat Model of Chronic Restraint Stress

Abstract Major depressive disorder (MDD) is strongly linked to chronic stress, yet its molecular mechanisms remain poorly understood. We hypothesize that chronic stress induces epigenetic alterations in stress-associated genes, contributing to transcriptional dysregulation in the prefrontal cortex (PFC), a key brain region implicated in MDD. To test this, we performed expression levels of stress-associated genes in the PFC of rats subjected to restraint stress, which revealed significant downregulation of CRHR2 and NR3C1 , both critical regulators of the hypothalamic–pituitary–adrenal (HPA) axis, as well as a trend to decreased expression of BDNF and TRKB , central mediators of synaptic plasticity. Given the well-established role of miRNAs in gene regulation, we further investigated stress-associated miRNA expression changes and identified miR-20b-3p and miR-425-3p as significantly upregulated in the restraint-stressed group. Bioinformatics analysis predicted miR-20b-3p as a potential regulator of NR3C1 , a relationship confirmed through RISC-mediated immunoenrichment, demonstrating increased miR-20b-3p binding to NR3C1 in stress-exposed rats. To confirm this interaction, we performed parallel analyses in cells ectopically expressing miR-20b-3p mimic, inhibitor, and a non-oligo control. Cells mimicking endogenous miR-20b-3p showed upregulated NR3C1 binding enrichment compared to non-transfected controls, while anti-miR-20b-3p treatment repressed its interaction with the NR3C1 3'UTR. These findings suggest that miR-20b-3p actively engages with NR3C1 and plays a regulatory role in its expression. Collectively, these results highlight a potential epigenetic mechanism by which chronic stress alters NR3C1 expression, possibly contributing to HPA axis dysregulation in MDD. The identification of miR-20b-3p as a key regulator of NR3C1 provides new insights into stress-induced molecular changes and suggests potential therapeutic targets for stress-associated psychiatric disorders.