The olive flounder (Paralichthys olivaceus) is one of the most consumed marine fishes in East and Southeast Asia. Streptococcosis is one of the major infectious diseases caused by Streptococcus sps. in humans, terrestrial and aquatic animals. In recent years, Streptococcus parauberis infections in olive flounder have increased and are severely affecting to their cultivation and economy. Extracellular vesicles (EVs) are heterogeneous group of cell-derived membranous structures with different cargos responsible for intercellular communication and homeostasis and been studied for their potentials in wound healing. MicroRNAs (miRNAs) are small, single-stranded, non-coding RNA molecules that regulate gene expression and are found in EVs. In this study, we focused on the wound healing properties of EVs derived from S. parauberis-infected olive flounders and their miRNA profiling for depth understanding of flounder EVs on wound healing and diverse other applications.
EVs were isolated from plasma samples of PBS-injected (PBS-Exo) and S. parauberis-challenged (Sp-Exo) olive flounders using ultracentrifugation method. Both EVs did not show significant in vitro and in vivo toxicity up to 400 μg/mL. The in vitro cell (human fibroblasts) migration assay, mRNA and protein expression results upon EVs treatment confirmed superior wound healing activity in Sp-Exo. The zebrafish larvae fin regeneration assay confirmed significantly higher tissue regeneration activity of Sp-Exo compared to PBS-Exo.
High-throughput miRNA profiling analysis of PBS-Exo and Sp-Exo illustrated that 14 known and 22 novel miRNAs were differentially expressed (DE) in Sp-Exo (log2 fold change >1 and <-1 Wald test p< 0.05). Pol-miR-21 and pol-miR-181 were identified as highly abundant miRNAs in Sp-Exo and are important for angiogenesis, fibroblast differentiation, and inflammatory reactions for supporting wound healing. GO enrichment and KEGG pathway analysis of DE miRNAs (hhi-miR-449, pol-miR-124, pol-miR-122 and pol-miR-203) revealed that their target genes contribute to various physiological and immunological functions, including wound healing and regeneration.
In conclusion, the miRNA profiling of Sp-Exo demonstrated potential features of wound healing activity. Further studies, such as proteomic analysis will support the development of Sp-Exo as a therapeutic agent to overcome limitations in aquaculture such as infection control and wound cure.
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) 2021R1A2C1004431.