Genome-Wide Analysis of MicroRNA-Messenger RNA Interactome in Ex-vivo Gill Filaments, Anguilla japonica

Gills of euryhaline fishes possess great physiological and structural plasticity to adapt to largechanges in external osmolality and to participate in ion uptake/excretion, which is critical forthe re-establishment of fluid and electrolyte homeostasis. The osmoregulatory plasticity ofgills provides an excellent model to study role of microRNAs (miRs) in adaptive osmoticresponses. The present study is to characterize an ex-vivo gill filament culture and usingomics approach, to decipher the interaction between tonicity-responsive miRs and genetargets, in orchestrating the osmotic stress-induced responses. Ex-vivo gill filament culturewas exposed to Leibovitz’s L-15 medium (300 mOsmol l–1) or the medium with an adjustedosmolality of 600 mOsmol l-1 for 4, 8 and 24 hr. Hypertonic responsive genes, includingosmotic stress transcriptional factor, Na+/H+exchange regulatory cofactor, with-no-lysinekinase, inward rectifying K+channel, cystic fibrosis transmembrane regulator, Na+/Cl--taurine transporter, Na+/K+-Atpase, and calcium-transporting ATPase were significantlyupregulated, while hypo-osmotic genes, like aquaporin-3 and V-type proton ATPase weredownregulated. The data illustrated that the ex-vivo gill filament culture exhibited distinctiveresponses to hyperosmotic acclimation, as compared with the current primary gill cell culturemodel. In the hyperosmotic treatment, four key factors (i.e. drosha RNase III endonuclease,exportin-5, dicer ribonuclease III and argonaute-2) involved in miR biogenesis weredysregulated. MicroRNA-sequencing of gill filament samples at 4 and 8 hr identified twodownregulated miRs, miR-29b-3p and miR-200b-3p. Integrated miR-mRNA-omics analysispredicted the functional implications of the miRs in calcium homeostasis, ion channelactivities and ATP production. Collectively, the ex-vivo gill filament culture would be auseful alternative model to investigate underlying mechanisms of osmotic responses in fishgills.