Comparison of physiological and biochemical indices
At present, research on the adaptability of Tibetan sheep to hypoxia at high altitude has made important progress in histology, morphology, physiology, and anatomy32,33,34,35. Studies suggest that compared to Tan sheep living in low-altitude areas, Tibetan sheep have developed a cerebral arterial system in which 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 and the regulation of cerebral arterial blood pressure36. This might be one of the anatomical characteristics of Tibetan sheep that aids in adaptation to a high-altitude hypoxic 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 fibres. These structural features are conducive to increasing alveolar ventilation, increasing pulmonary blood flow, accelerating blood oxygen transport, and improving the lung gas exchange rate in a hypoxic environment. Compared to low-altitude sheep, Tibetan sheep have more red blood cells and higher haematocrit and haemoglobin contents. Under a low oxygen environment, Tibetan sheep mainly adapt to a low oxygen environment by increasing the haemoglobin content of the blood37.
In this study, we first examined the haematological changes and serum biochemical parameters in four Tibetan sheep and one Hu sheep population. In agreement with previous reports, the haematological parameters, serum biochemical parameters, blood gas indices and 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 reason for the difference might be due to the extreme cold and hypoxic factors, especially the value of HGB, which increases with increasing altitude. Under low PO2, HGB dissociates from oxygen to provide the body with the oxygen needed for energy metabolism to better adapt to the low oxygen environment. Biochemical parameters, including AST, TP, ALB, GLO, ALP, and LDH, significantly increased with increasing altitude, while ALT and PCHE decreased with increasing altitude. In particular, the values of TP increased with altitude, which is helpful to enhance the immune function of sheep and adapt to the high-altitude ecological environment. Moreover, the blood gas indices, including PCO2, PO2, O2S, SBC, TCO2, and SBE, all significantly decreased with increasing altitude. Related studies have shown that Tibetan sheep can reduce tissue oxygen demand and cell metabolism levels through specific physiological changes and adapt to the plateau hypoxic environment at the molecular level by regulating hypoxia inducible factor13. Additionally, the morphology of lung tissue was observed, and we found that the terminal bronchioles, the number of alveoli counted per unit area, the alveolar septum thickness and the number of vessels per unit area significantly increased with increasing altitude. These changes in tissue structure are conducive to accelerating blood oxygen transport and increasing alveolar ventilation. This increases the lung blood flow and lung gas exchange rate of Tibetan sheep in a hypoxic environment to a certain extent. Early studies have shown that these changes are the key characteristics of adaptation to high-altitude environments by Tibetan sheep38,39.
Analysis of lncRNAs and their target genes
Due to the key roles of lncRNAs in many important biological processes, these are currently of particular interest40,41. The rapid development of high-throughput sequencing methods has led to the discovery of thousands of lncRNAs in recent years. Studies have reported that lncRNAs are involved in primary wool follicle induction in carpet wool sheep42, sheep fat-tail development43, sheep skeletal muscle development44, prolificacy in Hu sheep45 and sheep testicular maturation19,42 with high-throughput sequencing technology. However, the expression and function of lncRNAs in Tibetan sheep adaptation 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 transcript network that is involved in a high-altitude hypoxic environment.
The analysis of common DE genes among Tibetan sheep and Hu sheep found 2 common DE lncRNAs TCONS_00139593 and TCONS_00332125 in the liver and 1 common DE lncRNA TCONS_00377466 in the lung. The target genes of TCONS_00139593, including DOCK11, CYP2B4, TACO1, CYP2B11, ATP5SL, B3GNT8 BCKDHA, and EXOSC4, TCONS_00332125, including TTR and DSG3, TCONS_00377466, including WDR77, ZMYND19, and MRPL41, were found. Among them, DSG3, CYP2B4 and CYP2B11 are candidate genes related to high-altitude hypoxia adaptation, which are listed in Table 2. Moreover, the lncRNA–mRNA interaction network of liver samples 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 hub genes related to high-altitude hypoxia adaptation. The lncRNA–mRNA interaction network in lung samples 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 hub genes.
Preliminary research of candidate genes that are associated with hypoxia responses at high altitudes reported genome-wide scans that revealed positive selection in several regions that contained genes whose products are likely to be involved in high altitude adaptation46. Finally, a set of 247 functional candidate genes was identified. The functional candidate gene 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 the panther pathway. In this study, we found that target genes, including MB, PIK3R1, CYP1A1, MMP14, and TGFB1, belong to the list of 247 hypoxia genes. In addition, MMP1447, TUBB4B48, PSMD1349, COL3A1, COL1A250, DSG351,52, and ATP653 were also identified as candidate genes associated with high-altitude adaptation by previous functional studies.
Myoglobin, encoded by MB, is a haemoprotein present in cardiac, skeletal and smooth muscle that serves as a reserve supply of oxygen and facilitates the movement of molecular oxygen from the cell membrane to mitochondria54. A previous study demonstrated that PIK3R1, which is involved in the HIF-1α signalling pathway, plays a critical role in mediating adipose tissue insulin sensitivity55. Another previous study showed that CYP1A1 transcriptional activation was significantly decreased upon PCB 126 stimulation under conditions of hypoxia. Additionally, hypoxia pretreatment reduced PCB 126-induced AhR binding to CYP1 target gene promoters56. Additional research has shown that MMP14 is upregulated under hypoxic conditions and that this occurs by the interaction of HIF-1α and the MMP14 gene promoter region57. Chen et al. suggested that TGF-β1 encoded by TGFB1 decreases hypoxia–reoxygenation injury and attenuates alterations in NOS and PKB phosphorylation in myocytes exposed to hypoxia–reoxygenation58.
Yang et al.59 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, several positively selected genes within or regulating the HIF-1 pathway, the VEGF pathway, the VSMC pathway, glycolysis and lipids were identified 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)60. The detected SNPs were found in genes involved in angiogenesis, energy production and erythropoiesis and played a crucial role in hypoxia adaptation. Here, we found the target genes 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, which played a central role in regulating cellular responses to hypoxia46,61,62. Hypoxia regulates IGF1 expression through HIF-1α, and the inhibition of HIF-1α or IGF1R decreased CD133- and Oct4-positive GRPs under hypoxia63. Mora et al. found that the PDK1 signalling network plays an important role in regulating cardiac viability and preventing heart failure, and the deficiency of PDK1 in cardiac muscle results in heart failure and increased sensitivity to hypoxia64. 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, ischaemia and hypoxia65. Parra et al. found that the mRNA levels of the glycolytic markers HK2, PFKFB2 and GLUT1 increased in accordance with a metabolic shift towards nonmitochondrial ATP generation during hypoxia66. The VEGF pathway downstream of HIF-1 and glycolysis is an important mechanism of energy metabolism in 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.
The CYP2C31, CYP2B4, CYP2B5, and CYCS genes were functionally involved in oxygen binding, oxygen transport, and haem binding. In humans, indirect evidence suggests that hypoxia reduces the rate of biotransformation of drugs cleared by the cytochrome P450 subfamilies CYP1A, 2B, and 2C. Fradette et al. found that hypoxia downregulates rabbit hepatic CYP1A1, 1A2, 2B4, 2C5, and 2C16 and upregulates CYP3A6. CYP3A11 and P-glycoprotein were upregulated in the livers of hypoxic rats67. In addition, 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 the PSMD13 gene was associated with hypoxia by whole genome sequencing of lowland and highland chickens49. Qi et al. conducted a cross-tissue, cross-altitude, and cross-species study to characterize the transcriptomic landscape of domestic yaks. They found that the lung and heart are two key organs showing adaptive transcriptional changes, and five collagen genes (COL1A2, COL3A1, COL5A2, COL14A1, and COL15A1) highlight the crucial role of collagen-involved pathways in high-altitude adaptation50. Previous exome sequencing of five Chinese cashmere goat breeds revealed a candidate gene, DSG3, responsible for the high-altitude adaptation of the Tibetan goat. The mutations significantly segregated high- and low-altitude goats in two clusters, indicating the contribution of DSG3 to high-altitude hypoxia adaptation in Tibetan goats52. Wang et al. sequenced the ATP8 and ATP6 genes in 66 Tibetan yaks and 81 domestic cattle and found that haplotypes H4 in ATP8 and H5 in ATP6 present only in Tibetan yaks were suggested to be positively associated with high-altitude adaptation53.
Overall, the expression profile of lncRNAs and mRNAs in liver and lung tissue determined by the comparative transcriptome analysis between high- and low-altitude sheep indicates that the lung and liver are two key organs that show adaptive transcriptional changes. Moreover, the candidate genes involved in HIF-1, VEGF, and glycolysis/gluconeogenesis pathways, as well as oxygen binding, oxygen transport, and haem 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 index analyses, are valuable for understanding the genetic mechanism of hypoxic adaptation in sheep. Nevertheless, a limitation for this study is the earlier versions of the sheep reference genome was used.
