GEOapplication/xmlftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE338nnn/GSE338907/primaryOK200GenomicsTachysurus vachelliiNon-coding RNA profiling by high throughput sequencinghttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE338907GEOGSEfalseHepatic tissue-derived extracellular vesicles miRNA regulates hypoxia tolerance in Pelteobagrus vachellii by reprogramming metabolism to maintain liver homeostasisHypoxia is a critical environmental stressor that affects fish survival and physiological metabolism. Extracellular vesicles (EVs) mediate intercellular signal communication and perform vital functions in the stress response of aquatic organisms; however, the functional regulatory mechanisms of EVs under hypoxia in fish remain unelucidated. In this study, we optimized enzymatic digestion and combined with ultracentrifugation and high-precision iodixanol density gradient centrifugation, high-purity hepatic EVs were efficiently isolated from Pelteobagrus vachelli, which were internalized by primary hepatocytes successfully. Integrative analyses of the EVs miRNAome and hepatic transcriptome were performed to construct a miRNA-mRNA negative regulatory network, identifying metabolic reprogramming, HIF-1 signaling, cellular homeostasis and stress response as core pathways in hypoxic adaptation. In vivo functional validation revealed that hypoxia-induced EVs (Hypo-EVs) reduced the critical asphyxiation point, enhanced blood oxygen-carrying capacity, and alleviated hypoxia-induced hepatic oxidative damage in a dose-dependent manner, thereby significantly improving the hypoxia tolerance of P. vachelli. Mechanistic studies revealed that miR-135a is the core functional molecule within Hypo-EVs, and its expression was significantly downregulated under hypoxia. Overexpression of miR-135a reversed the hypoxic protective capacity of Hypo-EVs, whereas inhibition of miR-135a significantly enhanced hypoxia tolerance. Dual-luciferase and in vivo validation confirmed that miR-135a directly targets the 3'-UTRs of the glycolysis/gluconeogenesis key genes hk1 and pck2, negatively regulates glucose metabolism at the post-transcriptional level and mediates hypoxia adaptation through metabolic reprogramming. Collectively, this study clarifies the molecular mechanism by which the Hypo-EVs/miR-135a/hk1/pck2 signaling axis regulates hypoxia tolerance in P. vachellii. These findings provide theoretical references for research on fish hypoxic response mechanisms and stress-resilient aquaculture regulation.2026/07/21GSE338907GSM9884632GSM9884631GSM9884627GSM9884629GSM9884628GSM988463029689338907Tachysurus vachellii