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

Abstract

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.

Figure 1

In vivo expression and promoter ChIP assay for miR-218 gene. (A) miR-218a-5p expression was significantly higher in the CORT-treated group compared with the vehicle control group. Values denote mean ± SEM. U6 was used as normalizer. *p < 0.05. (B,C) Schematic diagram of miR-218a-1 and -2 locus and ChIP assay for GR binding on those promoter regions. miR-218a-3101 and -2 genes are located in the intronic regions of Slit2 and Slit3 respectively. We selected promoter regions (up to1k bp from TSS) which included predicted GR biding sites by in-silico for qPCR experiments. (D) There are increasing trends of GR biding in the CORT-treated group of Slit2, miR-218a-1 and miR-218a-2. (E) Significant enrichment of GR was found in the CORT-treated group of Slit3 promoter. *p < 0.05 and **p < 0.005. F, forward; R, reverse; GR, glucocorticoid receptor.

Figure 2

In vitro expression assay for miR-218-5p and RISC mediated target gene pulldown. (A) Relative expression profile of miR-218-5p in miR oligo transfected SH-SY5Y cells. Relative fold change analysis showed significantly higher expression of miR-218-5p in the mimic group compared to the vehicle group (t = 1.447, df = 3, p = 0.244). Values denote mean ± SEM. U6 was used as normalizer. *p < 0.005. (B) Volcano plot showing differential enrichment (up) and depletion (down) of target genes in RISC pull down (IP) complex. (C) Volcano plot showing differential expression profile of genes affected by miR-218-5p oligo overexpression based on input-RNA-seq results. (D) Six genes were detected as consistent target genes regulated by miR-218-5p overexpression. Consistent target genes were predicted by the following criteria: (1) significantly downregulated genes in the input RNA-seq and (2) significantly upregulated genes in the IP RNA-seq. In the chord diagram, the degree of interactions between input and IP groups on the six targets are shown with respective fold change values. The expression heat maps were created based on six genes with IP RNA-seq (E) and input RNA-seq (F).

Figure 3

Fold change of consistent target genes in the PFC of CORT-treated rats. Target gene expression differences between control and CORT treated rats were measured following relative fold change method. All five genes (Dtwd1, p = 0.019; Mettl22, p < 0.005; Snapc1, p < 0.001; Hdac6, p = 0.03) showed significant expression down regulation in CORT rat PFC except Bnip1 (p = 0.12) Values denote mean ± SEM. Gapdh expression values were used as normalizer. *p < 0.05 and **p < 0.005.

Figure 4

HDAC6-based ChIP-seq results in the PFC of CORT-treated rats. (A) Proportion of peak change regions in CORT treated rat PFC. (B) Ridgeline plots show the HDAC6-based chromatin peaks at the genomic scale. Chromatin peak distribution across genomic loci show close proximity to neighboring genes. The x-axis of the plot represents genes and Y-axis represents the distribution of HDAC6 associated chromatin peaks to map their distribution close to protein coding gene loci. (C) The HOMER based motif enrichment analysis identified the binding motif for ZNF264. (D) Connectivity of gene ontology (biological process) results based on nearest genes from the region of significant peak change.