Comparison of physiological and biochemical indices
At present, the research on the adaptability of Tibetan sheep to hypoxia at high altitude has made important progress in histology, morphology, physiology, and anatomy [31–34]. Studies suggest that compared to Tan sheep living at low altitude areas, Tibetan sheep have developed a cerebral arterial system, the main arteries are thicker in diameter, and the collateral branches in the cerebral arteries are developed and stretched longer. There are many small arteries, and this feature is conducive to effective blood supply to the brain tissue and the regulation of cerebral arterial blood pressure [35]. This might be one of the anatomical characteristics of Tibetan sheep aiding in adapting to high altitude hypoxia environment. Anatomical studies on the vascular system of other tissues and organs of Tibetan sheep have similar results. For example, compared to small-tailed Han sheep, Tibetan sheep have more capillaries in the alveolar septum, and they are mostly open, which also increases with altitude. The alveolar septum is thick, indicating that the alveolar septum is rich in capillaries and elastic fibers. These structural features are conducive to increasing alveolar ventilation, increasing pulmonary blood flow, accelerating blood oxygen transport, and improving lung gas exchange rate in a hypoxic environment. Compared to low-altitude sheep, Tibetan sheep have more red blood cells and higher hematocrit and hemoglobin content. Under low oxygen environment, Tibetan sheep mainly adapt to the low oxygen environment by increasing the hemoglobin content of the blood [36].
In this study, we first examined the haematological changes and serum biochemical parameters in four Tibetan sheep and a Hu sheep population. In agreement with previous reports, the haematological parameters, serum biochemical parameters, the blood gas indices and the morphology of lung tissues showed significant changes between Tibetan sheep (high altitude) and Hu sheep (low altitude). The haematological parameters including RBC, WBC, HGB, HCT, MCV, and PLT became significantly higher as the altitude increased (P < 0.05). The biochemical parameters including AST, TP, ALB, GLO, ALP, and LDH significantly increased with increasing altitude, while the ALT and PCHE decreased from increasing altitude. Moreover, the blood gas indices including PCO2, PO2, O2S, SBC, TCO2, and SBE all significantly decreased with increasing altitude. Additionally, the morphology of lung tissue was observed and we found that the terminal bronchioles, the number of alveolar counted per unit area, the alveolar septum thickness and the number of vessels per unit area significantly increased with increasing altitude.
Analysis of lncRNAs and their target genes
Owing to the key roles of lncRNAs in many important biological processes, these are currently of particular interest [37, 38]. The rapid development of high throughput sequencing methods had led to the discovery of thousands of lncRNAs in recent years. The studies have reported that the lncRNAs involved in primary wool follicle induction in carpet wool sheep [39], sheep fat-tail development [40], sheep skeletal muscle development [41], prolificacy in Hu sheep [42] and sheep testicular maturation [39, 43] with high throughput sequencing technology. But expression and function of lncRNAs in Tibetan sheep adapting to high altitude hypoxia are still unclear. To provide some insights into the biological functions of lncRNAs in Tibetan sheep adaption to high altitude hypoxia, a comprehensive analysis of lncRNA and mRNA profiling data from Tibetan sheep and Hu sheep, together with data from a public database was performed. We identified the core lncRNAs and their target genes, and validated their expression by qRT-PCR. Overall, our work uncovered an interlaced transcripts network that is involved in high altitude hypoxia environment.
By the analysis of common DE genes among Tibetan sheep and Hu sheep groups, 2 common DE lncRNAs TCONS_00139593 and TCONS_00332125 in liver and 1 common DE lncRNA TCONS_00377466 in lung were found. Moreover, the lncRNA–mRNA interaction network of liver sample showed that TCONS_00306477, TCONS_00306029, TCONS_00029720, TCONS_00145870, TCONS_00139593, TCONS_00380986, TCONS_00309307, TCONS_00225957, TCONS_00321529, and TCONS_00100469 interacted with more target genes and suggested as hub genes that related to high altitude hypoxia adaptation; the lncRNA–mRNA interaction network in lung sample showed that TCONS_00293272, TCONS_00313398, TCONS_00344932, TCONS_00078812, TCONS_00352306, TCONS_00380999, TCONS_00088235, TCONS_00467816, TCONS_00078180, and TCONS_00315164 interacted with more target genes and suggested as hub genes.
For the research of candidate genes that are associated with hypoxia responses at high altitudes, an early research reported genome-wide scans that revealed positive selection in several regions that contained genes whose products are likely to be involved in high altitude adaptation [22]. Finally, a set of 247 functional candidate genes were identified. The functional candidate genes categories included detection of oxygen (GO:0003032), NO metabolic process (GO:0046209), oxygen sensor activity (GO:0019826), oxygen binding (GO:0019825), oxygen transport (GO:0015671), oxygen transporter activity(GO:0005344), response to hypoxia (GO: 0001666), response to oxygen levels (GO:0070482), Vasodilation (GO:0042311), and hypoxia response via HIF activation (P00030) in panther pathway. In this study, we found that the target genes including MB, PIK3R1, CYP1A1, MMP14, and TGFB1 belong to the list of 247 hypoxia genes. Myoglobin, encoded by MB, is a haemoprotein present in cardiac, skeletal and smooth muscle and serves as a reserve supply of oxygen and facilitates the movement of molecular oxygen from the cell membrane to mitochondria [44]. The study has demonstrated that PIK3R1 involved in the HIF-1α signaling pathway plays a critical role in mediating adipose tissue insulin sensitivity [45]. Previous study shows CYP1A1 transcriptional activation was significantly decreased upon PCB 126 stimulation under conditions of hypoxia. Additionally, hypoxia pre-treatment reduced PCB 126 induced AhR binding to CYP1 target gene promoters [46]. Moore research showed that MMP14 is upregulated in hypoxic conditions and that this occurs by the interaction of HIF-1α and the MMP14 gene promoter region [47]. Chen et al suggested that TGF-b1 encoded by TGFB1 decreases hypoxia-reoxygenation injury and attenuates alterations in NOS and PKB phosphorylation in myocytes exposed to hypoxia-reoxygenation [48].
Yang et al [49] generated whole-genome sequences from 77 native sheep and detected a novel set of candidate genes as well as pathways and GO categories that were putatively associated with hypoxia responses at high altitudes. Specifically included several positively selected genes within or regulating the HIF-1 pathway, the VEGF pathway, the VSMC pathway, and glycolysis and lipid for energy metabolism. The network of relevant pathways indicated that hypoxia-induced factors, angiogenesis, vasodilatation and glycolysis metabolism were the most important factors that allowed sheep to manage extreme hypoxic environmental pressure. Seven sheep breeds representing both highland and lowland breeds from different areas of China were genotyped for a genome-wide collection of single-nucleotide polymorphisms (SNPs) [50]. Then detected selection events spanning genes were involved in angiogenesis, energy production and erythropoiesis played a crucial role in hypoxia adaption. Here, we found the target genes that PIK3R1, IGF1R and PDK1 in the classical HIF-1 pathway and FZD4, IFNB2, ATF3, PPCK1, PFKFB2 in the corresponding downstream VEGF and glycolysis/gluconeogenesis pathways. Hypoxia regulates IGF1 expression through HIF-1α, and the inhibition of HIF-1α or IGF1R decreased CD133- and Oct4-positive GRPs under hypoxia [51]. Mora et al found that the PDK1 signalling network plays an important role in regulating cardiac viability and preventing heart failure, the deficiency of PDK1 in cardiac muscle results in heart failure and increased sensitivity to hypoxia [52]. ATF3 is a stress-induced transcription factor that plays important roles in regulating immune and metabolic homeostasis. Overwhelming evidence confirms that the ATF3 gene is activated in many tissues by a variety of stress signals, including proinflammatory cytokines, ischemia and hypoxia [53]. Parra et al found that the mRNA levels of glycolytic markers HK2, PFKFB2 and GLUT1 increased accordance with a metabolic shift towards non-mitochondrial ATP generation during hypoxia [54]. The VEGF pathway downstream of HIF-1 and glycolysis is an important mechanism of energy metabolism for sheep under extreme hypoxic conditions. The dysregulation of genes in these pathways indicated that hypoxia-induced factors, angiogenesis, and glycolysis metabolism were the most important factors that allowed sheep to manage extreme hypoxic environmental pressure.
We also found the genes CYP2C31, CYP2B4, CYP2B5, and CYCS were functionally involved in oxygen binding, oxygen transport, and heme binding. In humans, indirect evidence suggests that hypoxia reduces the rate of biotransformation of drugs cleared by cytochrome P450 subfamilies CYP1A, 2B, and 2C. Fradette et al found that hypoxia down-regulates rabbit hepatic CYP1A1, 1A2, 2B4, 2C5, and 2C16 and up-regulates CYP3A6. CYP3A11 and P-glycoprotein were up-regulated in the livers of hypoxic rats [55]. In addition, the genes (TUBB4B, PSMD13, COL3A1, COL1A2, DSG3, and ATP6) were also identified as candidate genes associated with high altitude adaptation by previous functional studies. Kharrati-Koopaee et al found that PSMD13 gene was associated with the hypoxia by whole genome sequencing of lowland and highland chickens [26]. Qi et al conducted a cross-tissue, cross-altitude, and cross-species study to characterize the transcriptomic landscape of domestic yaks. They found that lung and heart are two key organs showing adaptive transcriptional changes, five of collagen genes (COL1A2, COL3A1, COL5A2, COL14A1, and COL15A1) highlighting the crucial role of collagen involved pathways in high altitude adaptation [27]. The previous exome sequencing of five Chinese cashmere goat breeds revealed a candidate gene, DSG3, responsible for the high altitude adaptation of the Tibetan goat. And the mutations significantly segregated high- and low-altitude goats in two clusters, indicating the contribution of DSG3 to the high altitude hypoxia adaptation in the Tibetan goat [29]. Wang et al sequenced the ATP8 and ATP6 genes in 66 Tibetan yaks and 81 domestic cattle found that haplotypes H4 in ATP8 and H5 in ATP6 present only in Tibetan yaks were suggested to be positively associated with high altitude adaptation [30].
Overall, the expression profile of lncRNAs and mRNAs in liver and lung tissue based on the comparative transcriptome analysis between high- and low- altitude sheep indicate that lung and liver are two key organs showing adaptive transcriptional changes. Moreover, the candidate genes involved in HIF-1, VEGF, and glycolysis/gluconeogenesis pathways, as well as oxygen binding, oxygen transport, and heme binding molecular function that were putatively associated with hypoxia responses at high altitudes were screened. These findings, in combination with the results of physiological and biochemical indices analysis, are valuable to understanding the genetic mechanism of hypoxic adaptation in sheep.