Through evolution, Tibetan chickens have developed genetic adaptability to survive in low-oxygen environments. Previous reports have shown that Tibetan chickens exhibit unique phenotypic and physiological characteristics, including enhanced blood oxygen binding capacity and increased blood circulation [11, 16]. Because it was first reported a few years ago, the ceRNA hypothesis has been widely accepted, leading to an improved understanding of human disease, as well as important advances in animal research [32, 34]. The recently discovered ncRNAs represent a new type of master regulator that affects gene expression and modulates a variety of cellular processes. Based on these theories, we constructed a ceRNA regulatory network in order to clarify the molecular mechanism of Tibetan chicken embryo CAM adaptation to hypoxic environments.
The CAM, an important respiratory and circulatory organ for chicken embryo development, contains a large number of blood vessels, and hypoxia can induce increased blood vessel density [17, 35]. From the constructed ceRNA network, we identified several miRNAs, including down-regulated novel-miR-589, novel-miR-815, novel-miR-85, novel-miR-669, as well as up-regulated miR-6606-5p, novel-miR-676, novel-miR-589, novel-miR-815, novel-miR-85, and novel-miR-567. Among these, miR-6606-5p was significantly up-regulated in the Tibetan chicken, and its target differentially expressed mRNAs (ACTC1, KCNMB4, and NCS1) were shown to be involved in vascular development under hypoxic conditions. ACTC1 was enriched in the GO term of blood circulation, while KCNMB4 has been shown to regulate blood pressure and was enriched in the GO terms of regulation of vasoconstriction, ion transport, and vascular smooth muscle contraction pathway . Previous studies also found that NCS1 regulates calcium ion transport and that Ca2+ is necessary for the activity of HIF-1, which is a major transcriptional regulator of cells and development in response to hypoxia [37, 38]. It is worth mentioning that with the exception of miR-6606-5p, other differentially expressed miRNAs, including miRNA-155, miRNA-302a, miR-302b-3p, miR-302b-5p, miR-302c-3p, miR-460b-3p, and miR-460b-5p, also play important roles in angiogenesis under hypoxia. The miRNA-155 promotes HIF-1α activity during prolonged hypoxia and participates in the PI3K/AKT pathway [39, 40]. Previous studies have shown that the miR-302 family (miR‐302a/b/c/d) suppresses the proliferation, migration, and angiogenesis of vascular endothelial cells by targeting VEGFA [41–43]. The miR-460b-3p and miR-460b-5p were identified in animal models of hypoxic pulmonary hypertension and were involved in the regulation of HIF-1α [44, 45]. Moreover, other differentially expressed mRNAs (NGFR, ADAM8, CASQ2, IRF4, PTPRZ1, CALML3, ERBB4, ARR3, and NTSR1) were enriched in blood vessel development, angiogenesis, blood circulation, hematopoiesis, response to hypoxia, oxygen transport, vascular smooth muscle contraction, calcium and MAPK signaling pathways. For example, NGFR is the receptor of NGF, which can induce chick CAM neovascularization . Studies have shown that ADAM8 is significantly induced by hypoxia and plays a role in the proliferation and migration of endothelial cells during angiogenesis [47, 48]. Therefore, we speculate that the enhanced tolerance of Tibetan chicken embryo CAM under hypoxic conditions is ascribed to miRNA-mediated modulation of the related target mRNAs, further regulating HIF, which enables it to maintain hypoxic adaptation via the promotion of angiogenesis and blood circulation.
Glucose uptake and carbohydrate metabolism are the basis for the maintenance of normal physiological functions in humans and animals. Under hypoxic conditions, oxygen and carbon dioxide metabolism mainly depend on mitochondrial respiration and make use of adenosine triphosphate (ATP). At present, a number of studies have shown the key regulatory role of energy metabolism, including glucose, carbohydrate, and lipid metabolic processes, among others, during hypoxic adaptation [49–52]. In the current research, from the constructed ceRNA network, we identified seven differentially expressed miRNAs (novel-miR-819, novel-miR-676, novel-miR-85, novel-miR-693, novel-miR-775, novel-miR-669, and novel-miR-867) and sixteen differentially expressed mRNAs related to energy metabolism including glycosylation, glucose metabolism process, carbohydrate metabolism process, fatty acid metabolism, and ATP binding. For example, SSTR5 and NR1H4 are essential for glucose homeostasis and play a pivotal role in glucose metabolism in animals [53, 54]. Cortisol regulates the metabolism of mouse adipose cells through the serotonin receptor gene HTR2C, and genetic variation of the APOA1 gene is linked to lipid metabolism and cardiovascular disease risk [55, 56]. It should be considered that more differentially expressed mRNAs (KCNB1, P3H2, CHST8, LYZ, HAO2, ACER1, ACSBG1, ELOVL2, ELOVL3, GBE, and NOX3) were targeted by key miRNAs, and several studies have shown that these mRNAs are also involved in energy metabolism. Therefore, we speculate that Tibetan chickens have enhanced energy metabolism due to the function of these RNAs, allowing for adaptation to hypoxic conditions.
Accumulating evidence indicates that lncRNAs play the role of ceRNAs (or miRNA sponges) in a variety of biological processes, including high-altitude adaptation. Such as LINC-PINT and LINC00599 polymorphisms are associated with high-altitude pulmonary edema in Chinese populations . Another study reported the expression profiles of lncRNAs in mice with high-altitude hypoxia-induced brain injury and provided new insights into the molecular mechanism of its treatment . In addition, a previous study reported the expression profiles of lncRNAs responsible for fatness and fatty acid composition traits in Tibetan pigs . In the current work, we found that the aforementioned differentially expressed mRNAs involved in angiogenesis and energy metabolism were targeted by 37 differentially expressed lncRNAs in the ceRNA network, suggesting that these lncRNAs may also function as miRNA sponges and may play a role in the hypoxic adaptation of Tibetan chicken embryos with regard to angiogenesis and energy metabolism.
Based on the ceRNA theory and the ceRNA network constructed in this study, we proposed the mode of action of differentially expressed lncRNAs, miRNAs, and mRNAs during hypoxic adaptation of Tibetan chicken embryos (Fig. 7). Under hypoxic conditions, miRNAs act as key regulators to modulate the up-regulation or down-regulation of important differentially expressed mRNAs. As a consequence, the angiogenesis/blood circulation of chorioallantoic capillaries and energy metabolism, such as glucose/carbohydrate metabolism, were stimulated, leading to the enhanced hypoxia adaptability of Tibetan chicken embryos. In this process, some lncRNAs act as ceRNAs to competitively bind the MRE of miRNAs, which may indirectly affect the expression of mRNA.