Data Sheet 1_MicroRNAs and genes regulating responses to hypoxia and inflammation expression levels in blood leukocytes as potential biomarkers of initial oxygen deficiency tolerance.pdf

IntroductionHypoxia-inducible factors (HIFs) play central roles in evoking responses to hypoxia, and their activities are regulated by numerous molecules, including microRNAs. Organisms typically show differing tolerances to oxygen deficiency; the most common method of determining such tolerance involves examining the effects of sublethal hypoxic exposure (SHE) in a decompression chamber, which can lead to pathological changes in the internal organs. The aim of the present study was to investigate the microRNAs and genes regulating cellular responses to hypoxia and inflammation expression levels in the peripheral blood leukocytes of laboratory animals before and 1 month following determination of hypoxia tolerance.
MethodsIn animals not exposed to hypoxic exposure, blood was collected from the tail vein. One month later, the rats’ resistance to hypoxia was determined at a critical altitude (11,500 m) in a single test in a decompression chamber, based on the “gasping time” before assuming a lateral position and the appearance of signs of asphyxia. Two groups of rats were identified – tolerant and susceptible to hypoxia. The expression of mRNA Hif1a, Epas1, Hif3a, Arnt, Vegf, Epo, Egln1, Nfkb, Il1b, Tnfa, Tgfb and microRNA rno-miR-210-5p, rno-miR-210-3p, rno-miR-107-5p, rno-miR-107-3p, rno-miR-145-5p, rno-miR-145-3p, rno-miR-155-5p, rno-miR-155-3p in leukocytes was determined by real-time PCR. To assess the impact of SHE on internal organs, morphological and morphometric studies of the lungs were carried out.
ResultsCompared to tolerant rats, the susceptible animals demonstrated an initial high proinflammatory potential characterized by high Hif1a, Epas1, Hif3a, and Nfkb expression along with low levels of rno-miR-155-3p and rno-miR-210-3p in the leukocytes. We observed the activation of proinflammatory responses in both tolerant and susceptible animals, and additional expression level increases of Il1b and Tnfa were noted only in the susceptible rats.
DiscussionSHE has extended effects on organisms that last for at least a month. The indicators identified herein, which differ between the hypoxia-tolerant and hypoxia-susceptible animals before sublethal hypoxic exposure, can therefore be considered as biomarkers of the initial hypoxia tolerance potential.

引言
缺氧诱导因子（Hypoxia-inducible factors, HIFs）在介导机体缺氧应答反应中发挥核心作用，其活性受包括微小RNA（microRNAs）在内的多种分子调控。生物体对缺氧的耐受能力通常存在差异；目前测定该耐受能力的最常用方法是在减压舱内开展亚致死性缺氧暴露（sublethal hypoxic exposure, SHE）实验，该操作可导致机体内部器官发生病理改变。本研究旨在探究缺氧耐受检测前后，实验动物外周血白细胞中调控缺氧与炎症细胞应答的微小RNA及基因的表达水平。

方法
在未接受缺氧暴露的实验动物中，通过尾静脉采集血液样本。1个月后，在减压舱内单次实验中，以动物侧卧前的喘息时间及窒息征象出现情况为依据，测定其在临界海拔（11500米）下的缺氧耐受能力。据此将大鼠分为缺氧耐受组与缺氧易感组。采用实时荧光定量PCR（real-time PCR）检测两组大鼠白细胞中缺氧诱导因子1α（Hif1a）mRNA、内皮PAS结构域蛋白1（Epas1）mRNA、缺氧诱导因子3α（Hif3a）mRNA、芳香烃受体核转位蛋白（Arnt）mRNA、血管内皮生长因子（Vegf）mRNA、促红细胞生成素（Epo）mRNA、EGL-1同源物1（Egln1）mRNA、核因子κB（Nfkb）mRNA、白细胞介素1β（Il1b）mRNA、肿瘤坏死因子α（Tnfa）mRNA、转化生长因子β（Tgfb）mRNA以及rno-miR-210-5p、rno-miR-210-3p、rno-miR-107-5p、rno-miR-107-3p、rno-miR-145-5p、rno-miR-145-3p、rno-miR-155-5p、rno-miR-155-3p的表达水平。为评估亚致死性缺氧暴露对机体内部器官的影响，本研究对肺部进行了形态学与形态计量学分析。

结果
与缺氧耐受组大鼠相比，缺氧易感组大鼠初始促炎潜能更高，表现为白细胞中缺氧诱导因子1α（Hif1a）、内皮PAS结构域蛋白1（Epas1）、缺氧诱导因子3α（Hif3a）及核因子κB（Nfkb）的表达水平升高，而rno-miR-155-3p与rno-miR-210-3p的表达水平降低。本研究观察到，耐受组与易感组大鼠均出现了促炎应答激活，但仅在易感组大鼠中检测到白细胞介素1β（Il1b）与肿瘤坏死因子α（Tnfa）的表达水平进一步升高。

讨论
亚致死性缺氧暴露对生物体的影响可持续至少1个月。因此，本研究鉴定出的、在亚致死性缺氧暴露前缺氧耐受组与易感组动物间存在差异的指标，可作为初始缺氧耐受潜能的生物标志物。