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Combined application of whole transcriptome sequencing and ceRNA theory: lncRNA LOC110519952 participates in glucose transport under hypoxia stress in rainbow trout (Oncorhynchus mykiss) by regulating novel-m0023-5p/glut1a axis

ABSTRACT: Hypoxia is a tricky and unavoidable environmental stressor for aquaculture with ongoing ecological deterioration and increasing stocking density, resulting in serious economic losses. Rainbow trout (Oncorhynchus mykiss), a commercially important fish species worldwide, exhibits extraordinarily poor tolerance to hypoxia; however, the regulatory mechanisms under hypoxia stress have not been elucidated. In this study, rainbow trout were separately subjected to moderate (Tm12L) and acute hypoxia for 12 h (Ts12L) and 12 h reoxygenation to investigate the changes of histology, biochemical parameters and transcriptomes (mRNA, miRNA and lncRNA) in liver, and interactions between LOC110519952-novel-m0023-5p-glut1a were further verified. The results showed that acute hypoxia resulted in more severe liver oxidative injury than moderate hypoxia, and anaerobic metabolism and lipid and protein metabolism were enhanced to some extent, especially in Ts12L. We also found that innate immune response was strengthened in Tm12L, while it was inhibited in Ts12L. In addition, dozens of hypoxia-related ceRNA networks derived from 1824 differentially expressed (DE) mRNAs, 427 DEmiRNAs and 545 DElncRNAs were identified, including LOC110519952-novel-m0023-5p-glut1a, LOC110520012-miR-206-y-vegfaa, LOC118965299-novel-m0179-3p-epoa and MSTRG.7382.2-miR-184-y-hmox. Further studies validated that LOC110519952 acted as a ceRNA that can positively regulate glut1a expression by adsorbing novel-m0023-5p. Overexpression of novel-m0023-5p could promote liver cell viability and proliferation, and introduction of LOC110519952 attenuated the effects. The findings contribute to deepen our understanding of the adaptive mechanisms used by rainbow trout in response to hypoxia stress, and provide valuable information for hypoxia-tolerant varieties breeding.

ORGANISM(S): Oncorhynchus mykiss

PROVIDER: GSE239701 | GEO | 2023/08/03

REPOSITORIES: GEO

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Project description: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.

Project description:The frequency of fish encountering hypoxia has risen dramatically as global warming and water pollution intensify, posing a serious threat to survival. Rainbow trout (Oncorhynchus mykiss), which is an economically important fish worldwide, belongs to a typical hypoxia-sensitive fish. However, little is known about the response mechanisms employed by rainbow trout to hypoxia stress. In this study, we systematically analyzed the changes of liver biochemical parameters in rainbow trout exposed to moderate hypoxic conditions (DO: 4.5 ± 0.1 mg/L) for different durations (4, 8, 12, 24 h and 1 month) and 12 h and 24 h reoxygenation, and histologic and transcriptome (lncRNA, miRNA and mRNA) under hypoxia stress for 12 h (Tm12L) and 1 month (TmlL) compared with control, and the interaction between LOC110520201, miR-2188-y and sod1 was verified by dual-luciferase reporter, overexpression and silencing assays. Histological observations revealed that liver suffered severe damage in Tm12L but not in TmlL. Integrating biochemical parameters and mRNA expression profiles, we found that exposure to short-term hypoxia stress resulted in enhanced immune response, aerobic and anaerobic glycolysis, gluconeogenesis and lipid metabolism. In TmlL, increased anaerobic glycolysis and decreased aerobic glycolysis were observed, and antioxidant and immune capacity has returned to normal. Additionally, several key hypoxia-related genes (epo, vegfc, epor, sod1, ppara, foxo1a, gk, dusp1, nlrc3 and nlrp3) and ceRNA regulatory networks were identified from 2711 differentially expressed (DE) mRNAs, 575 DElncRNAs and 395 DEmiRNAs, including LOC110520201-miR-2188-y-sod1, LOC110533332-miR-144-y-vegfc, LOC110526066-miR-465-x-ppara and LOC110535032-miR-743-y-nlrp3. The analysis of expression patterns in liver and rainbow trout liver cells treated with hypoxia suggested that changes in LOC110520201, miR-2188-y and sod1 showed a ceRNA regulatory relationship. Further experiments demonstrated that sod1 is a target of miR-2188-y, and LOC110520201 silencing increased the expression of miR-2188-y and inhibited sod1 expression. These results facilitated our understanding of the molecular mechanisms of hypoxia response in rainbow trout and provided genetic resources for breeding hypoxia-tolerant varieties.

Dataset Information

Combined application of whole transcriptome sequencing and ceRNA theory: lncRNA LOC110519952 participates in glucose transport under hypoxia stress in rainbow trout (Oncorhynchus mykiss) by regulating novel-m0023-5p/glut1a axis

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