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.