Corticosterone-mediated regulation and functions of miR-218-5p in rat brain

Chronic stress is one of the key precipitating factors in major depressive disorder (MDD). Stress associated studies have underscored the mechanistic role of epigenetic master players like microRNAs (miRNAs) in depression pathophysiology at both preclinical and clinical levels. Previously, we had reported changes in miR-218-5p expression in response to corticosterone (CORT) induced chronic stress. MiR-218-5p was one of the most significantly induced miRNAs in the prefrontal cortex (PFC) of rats under chronic stress. In the present report, we have investigated how chronic CORT exposure mechanistically affected miR-218-5p expression in the rat brain and how miR-218 could trigger molecular changes on its downstream regulatory pathways. Elevated expression of miR-218-5p was found in the PFC of CORT-treated rats. A glucocorticoid receptor (GR) targeted Chromatin-Immunoprecipitation (ChIP) assay revealed high GR occupancy on the promoter region of Slit3 gene hosting miR-218-2 in its 3rd intron. RNA-sequencing data based on RNA Induced silencing Complex Immunoprecipitation (RISC-IP) with AGO2 in SH-SY5Y cells detected six consistent target genes of miR-218-5p (APOL4, DTWD1, BNIP1, METTL22, SNAPC1, and HDAC6). The expression of all five genes, except APOL4, was successfully validated with qPCR in CORT-treated rat PFC. Further, Hdac6-based ChIP-seq experiment helped in mapping major genomic loci enriched for intergenic regions in the PFC of CORT-treated rat. A proximity-based gene ontology (GO) analysis revealed a majority of the intergenic sites to be part of key genes implicated in central nervous system functions, notably synapse organization, neuron projection morphogenesis, and axonogenesis. Our results suggest that the upregulation of miR-218-5p in PFC of CORT-treated rats possibly resulted from GR biding in the promoter region of Slit3 gene. Interestingly, Hdac6 was one of the consistent target genes potentially found to regulate CNS related genes by chromatin modification. Collectively, these findings establish the role of miR-218-5p in chronic stress and the epigenetic function it plays to induce chromatin-based transcriptional changes of several CNS genes in triggering stress-induced disorders, including depression. This also opens up the scope to understand the role of miR-218-5p as a potential target for noncoding RNA therapeutics in clinical depression.