Bioinformatic Discovery of Novel MicroRNAs in Citrus sinensis Extracellular Vesicles and Comparative Analysis with Human MicroRNAs

MicroRNAs (miRNAs) found in plant-derived extracellular vesicles (EVs) show promise as therapeutic agents due to their potential ability to regulate gene expression across kingdoms. This study investigates miRNAs in Citrus sinensis EVs and their possible therapeutic applications in wound healing. Using miRDeep2, the identification and characterization of both known and novel miRNAs derived from citrus EVs isolated through different extraction methods was performed. The analysis revealed that RNA extraction methodology significantly influences miRNA detection compared to EV isolation methods and the source of the sample. Through sequence alignment analysis, we identified 12 citrus miRNAs showing significant similarity to human miRNAs, particularly in their seed regions. Overlapped target prediction and pathway analysis of citrus-human miRNA pairs revealed enrichment in key wound healing pathways, including PI3K-Akt and MAPK signaling. Unexpectedly, the enrichment was found in neuronal-related processes, suggesting potential roles in nerve regeneration during wound healing. Integration with wound healing transcriptome data demonstrated a possible miRNA-mRNA axis, providing potential regulation patterns at different healing stages, involving growth factors such as TGFBR1, FGF2, and ANGPT2. Our findings provide novel insights into the therapeutic potential of citrus EV-derived miRNAs and establish a bioinformatic foundation for developing plant-based therapeutics for wound healing. However, experimental validation of these predicted interactions and further investigation of EV-mediated miRNA delivery mechanisms are needed to advance these findings toward clinical applications.