Integrated RNA-seq and functional analyses reveal the potential role of microRNAs in rainbow trout (Oncorhynchus mykiss) adapting to acute hypoxia and reoxygenation stress
Hypoxia negatively affects the behavior, growth, reproduction, and survival of fish, causing serious economic losses to aquaculture. Rainbow trout (Oncorhynchus mykiss), an important economic fish worldwide, belongs to a hypoxia-sensitive fish species. However, the regulatory mechanisms of miRNAs under hypoxia stress response of rainbow trout remains unclear. In this study, rainbow trout were subjected to hypoxia stress (DO: 3.5 mg/L) for 3 h (H3h_L), 12 h (H12h_L), 24 h (H24h_L) and 3 h reoxygenation (R3h_L) to systemically evaluate the changes of miRNA expression profiles in liver, and the functions of sha-miR-92a_L+2R+4 were investigated. We found 17, 144, 57 and 55 differentially expressed (DE) miRNAs in H3h_L vs. control (N_L), H12h_L vs. N_L, H24h_L vs. N_L and R3h_L vs. N_L comparisons, respectively. Enrichment analysis revealed that the targets of these DE miRNAs were significantly enriched in HIF signaling pathway, VEGF signaling pathway, FoxO signaling pathway and glycolysis/gluconeogenesis. Through miRNA-mRNA nteraction and weighted gene co-expression network analysis (WGCNA), five key DE miRNAs (sha-miR-92a_L+2R+4, ssa-miR-128-3p, ssa-miR-101b-3p_R+1, ola-miR-199a-5p_R+2 and tni-miR-199_1ss18CG) were identified, which can target at least two hypoxia-responsive genes, such as vegfaa, ho, glut1a and junb. Functional analysis found that sha-miR-92a_L+2R+4 directly regulated vegfaa expression by targeting its 3'-UTR, overexpression of sha-miR-92a_L+2R+4 significantly decreased vegfaa expression in rainbow trout liver cells, while opposite results were obtained after transfection of sha-miR-92a_L+2R+4 inhibitor. Furthermore, overexpression of sha-miR-92a_L+2R+4 promoted rainbow trout liver cell proliferation and inhibited apoptosis. These results deepen our understanding of the crucial roles of miRNAs under hypoxia stress in rainbow trout, and provide valuable information for further studying the regulatory mechanisms of key hypoxia-responsive miRNAs and breeding hypoxia-tolerant rainbow trout species. Overall design: In the present study, the miRNA expression profiles changes in the liver of rainbow trout under hypoxia for different durations and reoxygenation stress were detected using the Illumina HiSeq 2500 sequencing platform, and the functions of sha-miR-92a_L+2R+4 were further investigated.