Exosomal miRNA Profiling in Liquid Biopsy of Vitreous in Proliferative Diabetic Retinopathy

Purpose: Proliferative diabetic retinopathy (PDR) is a severe microvascular complication of diabetes mellitus. Although exosomes participate in various pathologic processes, their role in ocular pathologies is unclear. This study profiled exosomal microRNAs (exo-miRNAs) in the vitreous humor (VH) of patients with PDR to elucidate their regulatory roles in PDR pathogenesis. Methods: VH samples were obtained from 23 patients with PDR and 17 non-diabetic controls. Exosomes were isolated using ultracentrifugation and characterized via transmission electron microscopy, nanoparticle tracking analysis and Western blot. High-throughput sequencing identified differentially expressed miRNAs (DEMs), followed by target gene prediction and bioinformatic analyses. The top DEMs were validated using quantitative real-time polymerase chain reaction (qRT-PCR). Results: Exosomes were isolated and characterized from VH samples. Sequencing analysis identified 843 unique miRNAs, with 60 showing significant differential expression between PDR and control groups (28 upregulated and 32 downregulated). The miR-451a and miR-486-5p were most upregulated; miR-204-5p and miR-211-5p were most downregulated. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed DEM target genes enriched in endocrine, metabolic, signaling transduction, and intercellular adhesion pathways, notably cGMP-PKG, mTOR, and cAMP pathways. Network analysis identified miR-486-5p and miR-451a as key hubs. Validation by qRT-PCR confirmed the differential expression in the sequencing analysis. Conclusions: This study provides the comprehensive profile of vitreous exo-miRNAs in patients with PDR, identifying key DEMs and their potential regulatory networks. These findings indicate that vitreous exo-miRNAs, specifically miR-204-5p and miR-486-5p, represent promising diagnostic biomarkers and potential therapeutic targets for PDR.