Multi-omics Analysis And HypoxamiR Related Mechanism Of The Effects Of Hypoxia On Mouse Brain Microvascular Endothelial Cells

With the development of economy,the number of people from the plains traveling to the plateau for short-term and long-term stays is increasing.Although high-altitude environments pose challenges such as cold,dry conditions,and intense ultraviolet radiation,the most critical health concern faced is hypobaric hypoxia induced by changes in atmospheric pressure.Previous research indicated that acute exposure to high-altitude environments（>2500 m）may lead to acute high-altitude illnesses（AHI）.Acute mountain sickness（AMS）and high-altitude cerebral edema（HACE）are closely related to the central nervous system（CNS）.The increase in blood-brain barrier permeability and dysfunction of brain microvascular endothelial cells are considered as important factors in the occurrence and development of AHI.However,the molecular mechanisms are not fully understood.At the same time,there is a lack of effective intervention and protective measures.Micro RNA（miRNA）is an endogenous noncoding RNA with approximately 18-22nucleotides.It can induce m RNA degradation or inhibit translation by binding to the 3’untranslated region（3’UTR）of their target genes,playing an important regulatory role in animals and plants.miRNA can potentially regulate various aspects of cellular function,including proliferation and differentiation,energy metabolism,DNA repair,infection,immunity,cell death,etc.Previous studies showed that hypoxia upregulates the expression of specific miRNAs,known as hypoxia induced miRNAs or"hypoxamiR",involved in the cellular response to oxygen level.However,the role and mechanism of hypoxamiR in BMEC under hypoxia are not fully elucidated.This study aimed to reveal the molecular and functional changes of BMEC under hypoxia through multi omics,and further explore the role and mechanism of hypoxamiR.Objectives1.To explore the effects of locomotor activity and BMEC in mice under acute hypoxia.2.To reveal the molecular change profile of BMEC under hypoxia through multi-omics analysis.3.To explore the effects of cell cycle and proliferation of BMEC under hypoxia,as well as the role and mechanism of hypoxamiR.4.To explore the effects of the mitochondrial function and apoptosis of BMEC under hypoxia,as well as the role and mechanism of hypoxamiR.MethodsThe open field test（OFT）was used in large hypobaric chamber to explore the effects of locomotor activity in mice under hypoxia.Multi-omics and enrichment analysis were used to clarify the effects of hypoxia on cellular signaling pathways and biological processes.RT-q PCR,transmission electron microscopy,western blot,immunofluorescence staining,flow cytometry,JC-1 staining,Ed U staining,CCK8 assay,FISH,dual-luciferase reporter gene assay,lentivirus,rapid amplification of c DNA end and Ch IP assay were used to investigate the role and mechanism of hypoxamiR in BMEC under hypoxia.Results1.Acute hypoxia significantly affected results of open field tests in mice;as the altitude increases,the impact gradually intensifies,but there are no results indicating a correlation with exposure time within 24 h at 5000 m;the v WF and CD31 positive cells in the brain of mice exposed to 5000 m for 24 h were reduced.2.The RNA,protein,and metabolite levels of BMEC was significantly changed under hypoxia（1%O₂）for 24 h;the HIF-1 signaling pathway,cell cycle,DNA replication,energy metabolism,glucose metabolism,protein synthesis,nc RNA processing,inflammation and other signaling pathways of BMEC were significantly changed;the cell cycle and DNA replication were significantly downregulated,while the glycolysis/gluconeogenesis and HIF-1 signaling pathways were significantly activated.3.1%O₂ hypoxia for 24 h inhibited cell cycle progression and cell proliferation in BMEC;miR-212-3p level was significantly increased under hypoxia;miR-212-3p directly targeted Mcm2 3’UTR and downregulated the expression of MCM2;miR-212-3p inhibited BMEC cell cycle and proliferation,and miR-212-3p inhibitor attenuated the inhibition of cell cycle progression and cell proliferation caused by hypoxia;knockdown of Mcm2inhibited BMEC cell cycle and proliferation;Mcm2 overexpression attenuated hypoxia-induced inhibition of cell cycle and cell proliferation;hypoxia regulated the cell cycle progression and proliferation of BMEC via the miR-212-3p/MCM2 axis.4.Hypoxia damaged the mitochondrial structure and function and increased ROS and apoptosis levels of BMEC;1%O₂ hypoxia for 24 h increased the level of miR-210-3p;miR-210-3p reduced the mitochondrial membrane potential of BMEC and increased ROS and apoptosis levels;miR-210-3p inhibitor attenuated the decrease in mitochondrial membrane potential and the increase in ROS and apoptosis levels caused by hypoxia;miR-210-3p directly targeted the 3’UTR of Iscu and Ndufa4 to downregulate their expression;HIF1A promoted miR-210-3p expression by binding to the miR-210 promoter;hypoxia through HIF1A/miR-210-3p regulating ISCU and NDUFA4 is highly conserved in many organisms.Conclusions1.Acute hypoxia significantly affected the locomotor activity and mouse brain microvascular endothelial cells of mice.2.The cell proliferation,energy metabolism,nc RNA processing pathways and hypoxamiR levels of BMEC were significantly affected under hypoxia.3.Hypoxia inhibited the cell cycle progression and proliferation of BMEC through the miR-212-3p/MCM2 axis.4.Hypoxia induced mitochondrial damage and apoptosis through HIF1A/miR-210-3p regulating ISCU and NDUFA4.This pathway is highly conserved in many organisms.