microRNA-29b knocks down collagen type I production in cultured rainbow trout (Oncorhynchus mykiss) cardiac fibroblasts.

Warm acclimation of rainbow trout can cause a decrease in the collagen content of the heart. This ability to remove cardiac collagen is particularly interesting considering that collagen deposition in the mammalian heart, following an injury, is permanent. We hypothesized that collagen removal can be facilitated by microRNA-29b (miR-29b), a highly conserved, small, non-coding RNA, as a reduction in this microRNA has been reported during the development of fibrosis in the mammalian heart. We also used a bioinformatics approach to investigate the binding potential of miR-29b to the seed sequences of vertebrate collagen isoforms. Cultured trout cardiac fibroblasts were transfected with zebrafish mature miR-29b mimic for 7 days with re-transfection occurring after 3 days. Transfection induced a 17.8-fold increase in miR-29b transcript abundance (P<0.05) as well as a 54% decrease in the transcript levels of the col1a3 collagen isoform, compared with non-transfected controls (P<0.05). Western blotting demonstrated that the level of collagen type I protein was 85% lower in cells transfected with miR-29b than in control cells (P<0.05). Finally, bioinformatic analysis suggested that the predicted 3'-UTR of rainbow trout col1a3 has a comparatively higher binding affinity for miR-29b than the 3'-UTR of col1a1 Together, these results suggest that miR-29b is a highly conserved regulator of collagen type I protein in vertebrates and that this microRNA decreases collagen in the trout heart by targeting col1a3.

Diploid and triploid Chinook salmon (Oncorhynchus tshawytscha) have altered microRNA responses in immune tissues after infection with Vibrio anguillarum.

Production of sterile fishes through artificial retention of a third set of chromosomes (triploidy) is a sustainable alternative for aquaculture since it reduces escapee pressure on wild populations. However, these fishes have reduced survival in stressful conditions and in response to infection. In this study, the impact of Vibrio anguillarum infection on diploid and triploid Chinook salmon (Oncorhynchus tshawytscha) was investigated to identify if there was any significant immune regulation by microRNAs (miRNA). Small RNAs from hindgut, head kidney, and spleen were sequenced to determine if miRNA transcript abundance was altered due to ploidy and infection in nine-month old full-sibling diploids and triploids. All three tissues had differentially expressed miRNA prior to infection, indicating subtle changes in epigenetic regulation due to increased ploidy. Additionally, miRNA were altered by infection, but there was only a difference in spleen miRNA expression between diploids and triploids at three days of infection. Furthermore, one miRNA (ssa-miR-2188-3p) was confirmed as having an altered response to infection in triploids compared to diploids, implicating potential immune dysregulation due to increased ploidy. The miRNAs identified in this study are predicted to target immune pathways, providing evidence for their importance in regulating responses to pathogens. This study is the first to investigate how increased ploidy alters miRNA expression in response to infection. Additionally, it provides evidence for epigenetic dysregulation in triploid fishes, which may contribute to their poor performance in response to stress.

Acute air exposure modulates the microRNA abundance in stress responsive tissues and circulating extracellular vesicles in rainbow trout (Oncorhynchus mykiss).

The hypothalamic-pituitary-interrenal axis is an important regulator of stress and metabolism in teleosts. Cortisol is secreted by the head kidney where it increases gluconeogenesis in the liver to increase circulating glucose levels. MicroRNAs (miRNAs) are small, non-coding RNA molecules that bind to the 3' untranslated region of specific mRNA to regulate their expression. MicroRNAs can also be secreted into circulation by association with extracellular vesicles (EVs) where they can influence the phenotype of other tissues. In this study, adult rainbow trout were exposed to a 3-minute acute air stress and allowed to recover for 1-, 3-, or 24-h to determine how miRNAs were altered. MicroRNAs measured in this study were chosen based on their high relative abundance in tissues that drive the stress response (miR-21a-3p, let-7a-5p, miR-143-3p) or their role in regulating DNA methylation (miR-29a-3p). In general, miRNAs increased in circulating EVs during the recovery period while decreasing in head kidney and liver at the same timepoints. Predicted targets for these miRNAs were analyzed using KEGG and DAVID functional enrichment analysis. Pathways involved in metabolism and cell signaling were predicted to be upregulated. Future studies can use these results to investigate how pathways are regulated after stress. Overall, our results indicate that miRNAs are regulated during teleost stress responses and could be supporting the cortisol-mediated changes that occur.