Profiling fear-extinction learning induced small ncRNAs landscape and identifying miR-29b-3p as a promising biomarker for PTSD

The pivotal role of small non-coding RNAs (ncRNAs) in gene expression highlights their potential as therapeutic targets and biomarkers in neuropsychiatric disorders. However, their transcriptional dynamics during fear extinction (EXT) remain poorly understood, limiting insights into disorders such as post-traumatic stress disorder (PTSD). We profiled the small ncRNAs transcriptome in the medial prefrontal cortex (mPFC) following EXT training using small RNA array and explored the potential biological functions of differentially expressed (DE) miRNAs. Publicly available miRNA expression data from peripheral blood mononuclear cells (PBMCs) of PTSD patients was integrated to identify conserved miRNAs biomarkers. Validation of the expression of candidate miRNA in mouse mPFC, plasma and PTSD patients’ PBMCs were performed by RT-qPCR. Inhibitor of candidate miRNA was infused into the infralimbic prefrontal cortex (ILPFC) of mice to assess its effect on fear extinction memory. To assess the impact of Dnmt3a, a common candidate hsa- and mmu-miRNA target, on DNA methylation, publicly available whole-genome bisulfite sequencing (WGBS) data from Dnmt3a knockout or mutant mice across four brain regions were analyzed. Based on the small RNA data, we also analyzed DE tsRNAs, including their subtype distributions, tRNA origins, and potential functions. We identified 63 DE miRNAs following EXT training, with predicted targets enriched in dendritic and synaptic function. In PTSD patient PBMCs, 250 DE miRNAs were identified, including three conserved extinction-induced miRNAs that were downregulated in PBMCs, which were further confirmed in our mouse model and PTSD human blood samples. We next demonstrated that miR-29b-3p facilitates the formation of fear extinction memory. Bioinformatic analysis suggests that miR-29b-3p targets the 3’ untranslated region of Dnmt3a mRNA, which encodes a DNA methyltransferase involved in de novo non-CG methylation in brain. For other small ncRNAs, we identified 75 DE tsRNAs, with 5′tiRNA and 3′tiRNA being the most abundant in the up-regulated and down-regulated groups, respectively. Gln-CTG, Lys-CTT and Lys-TTT was identified as the predominant tRNA contributing to the biogenesis of DE tsRNAs. Our findings emphasize the critical role of small ncRNA regulation in fear extinction learning and highlight miR-29b-3p as a promising functional biomarker for PTSD.