Genomics and Bioinformatics Enhance the Study of HAPC
Upon completing the Human Genome Project, we will have a more comprehensive understanding of population-specific genomic variations and the interactions between genes and environmental factors. This information will help us quickly determine mutation sites related to diseases to use genetic information to establish the relationship between sequence variation and disease risk and help prevent, diagnose, and treat diseases [18, 19].
HAPC seriously intimidates the fitness of the population of the plateau. In Tibet, the increased Hb concentration enhances the efficiency of carrying oxygen to adapt to altitude hypoxia. This reaction is an imperative factor for populations, who adapt to high altitudes. We found an increased risk of HAPC associated with SNPs in EPO, STAT3, PDK1, STAT5A, IL12RB1, PTK2, MET, TCL1A, RELN, RUNDC3B, TDRD1, and NF2 in the Tibetan population. We found that these genes were mostly enriched in the PI3K-AKT, JAK-STAT, and HIF-1 pathways through KEGG pathway analysis.
Notably, the genes' distributions varied widely from the different pathways based on the KEGG pathway analysis. From the perspective of the pathways, three genes EPO, STAT3, and PDK1 were enriched in HIF-1 pathways; four genes, EPO, STAT3, STAT5A, and IL12RB1, were enriched in JAK-STAT pathways; five genes EPO, PTK2, MET, TCL1A, and RELN were enriched in PI3K-AKT pathways. Likewise, EPO gene was enriched in these three pathways; STAT3 gene was enriched in two pathways; excluding RUNDC3B, TDRD1, and NF2 genes, other genes were positioned in one pathway.
The Impact of Genes Enriched in the HIF-1 Pathway for Erythrocytes
Hypoxia-inducible factor one (HIF-1), as a transcription factor, is composed of two subunits, which include an induced expressed part of HIF-1α subunit and a control structure of HIF-1β subunit. Without hypoxia, HIF-1α subunit would never be ubiquitinated and hydrolyzed by proteasomes easily . Meanwhile, complex regulation of signal transduction cascades, which are mediated by cytokines and their homologous receptors, may affect the formation of HAPC. When the body is in a low-oxygen environment, its red blood cells increase. The HIF-1 signaling pathway may activate this.
EPO is the earliest hypoxic adaptation gene, and its polymorphism is related to the formation of HAPC . Our results conformed these findings as we found that the rs773485910 of the EPO gene was associated with an increased prevalence of HAPC. EPO, endogenous glycoprotein hormone, has a molecular weight of 39 KD, with 166 residues. It is a member of the hematopoietic cytokine family and is primarily involved in erythropoiesis. EPO is principally secreted by liver cells in infancy and by kidney cells in adulthood. When the body immerses in a low-oxygen environment, the acetylation level of HIF-1 alpha increase, and there is enhanced transcriptional activation of a series of target genes, stimulating increased secretion of EPO by the kidneys . Via binding to the EPO receptor (EPOR) on red cell progenitors in the bone marrow, circulating EPO can arouse an increase in the amount of erythrocytes . However, small amounts are also expressed in other vital organs, such as the brain, spleen, lungs, testicles, and placental tissue. At average oxygen concentrations, the EPO content of blood is low, and its primary role relates to the renewal of aging erythrocytes .
PDKs are serine protein kinase genes located on chromosome 2, mainly expressed in the heart, bone marrow, kidneys, and skin. There are four isomers of PDK–PDK1, PDK2, PDK3, and PDK4. PDK1, PDK2, and PDK3 are mainly involved in cellular glucose metabolism, while PDK4 is engaged primarily in cell fat metabolism. Previous studies have shown that PDK1 and PDK4 genes are associated with erythrocyte polymorphism in Chuvash decent people. Our research found that rs529091195 of the PDK gene was associated with HAPC, which might be caused by significant upregulation of HIF due to chronic hypoxia. Upregulated HIF can promote PDK1 expression, affecting the anaerobic oxidation of cells and providing energy for normal cellular life activities .
Moreover, by retrieving PDK1 in the KEGG pathway database, we acquire that PDK1 also brings a crucial impact in the PI3K-AKT pathway and is activated by plasma membrane intrinsic protein 3 (PIP3). Activated PDK1 fully activates adjacent protein kinase B (AKT) and regulates downstream AGC family protein kinase activity [25,26,27]. These actions allow PDK1 to control the physiological effects of insulin and growth factors, increase glucose uptake, promote glycogen and protein synthesis, and provide energy for cell proliferation and differentiation . In this way, PDK1 influences the development of HAPC. Once activated, the PI3K-AKT pathway can also play an anti-apoptotic role, resulting in increased erythrocyte accumulation and promoting HAPC .
The Impact of Genes Enriched in the JAK-STAT Pathway for Erythrocytes
Important hematological factors widely involve the JAK-STAT pathway. When EPO binds to the EPO receptor, the JAK-STAT signaling pathway can also be activated . Among those principal kinases involved in mediating EPO-responsive signal transduction, the Jak2 protein tyrosine kinase was identified for the first time by the researchers. JAK2 binds to EPOR at the bottom of the cytoplasm, causing JAK2 phosphorylation, which leads to tyrosine phosphorylation and coupling of STAT-5, affecting cell proliferation and differentiation .
STATs consist of seven separate members, STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6. Furthermore, they are active in many cell signaling pathways and have essential effects on innate immunity, acquired immunity, cell proliferation, differentiation, and survival . We found that rs558351915 of the STAT3 gene and rs779456792 of the STAT5A gene were related to the formation of HAPC. Previous studies have shown that different STAT family members can interact in cell signaling pathways to regulate target genes' expression . The JAK-STAT pathway plays an imperative role in cellular erythropoiesis, proliferation, and differentiation. STAT3 and STAT5 are essential phosphorylated kinases in the JAK-STAT cell signaling pathway. Meanwhile, EPO binds to the EPO receptor to activate and phosphorylated JAK2; and tyrosine phosphorylated and coupled with STAT5. Then, JAK1 and STAT5 upregulate the expression of membrane proteins, cytoskeleton, hematopoietic growth factor-related genes, and downstream target genes, which contain anti-apoptotic genes Bcl-xl and Bcl-2. Moreover, the PI3K-AKT pathway is a downstream effector of JAK2-STAT5 signaling. Also, STAT5 regulates and promotes hemoglobin expression, thereby induces the proliferation of red blood cells (RBC). In a word, it can increase the RBCs' count in the body's blood .
The Impact of Genes Enriched in the PI3K-AKT Pathway for Erythrocytes
The PI3K-Akt signaling pathway is also called the "cell survival signaling pathway," which can protect cells from inactivation and affect cell proliferation and differentiation by being activated. This signaling pathway brings a substantial impact on the process of erythropoiesis and could downregulate apoptosis by regulating apoptosis-related molecules. This process promotes the expression of the anti-apoptotic gene Bcl-xL and plays a critical anti-apoptotic role . In particular, PI3K-AKT pathways affect HIF-1α transcriptional activity in hypoxia [35, 36], sequentially transactivating EPO, eventually resulting in erythrocytosis. Therefore, the PI3K-AKT signal pathway appeared to be involved in the mechanism of decreased erythroblasts apoptosis.
PTK2 genes are enriched in the PI3K-AKT signaling pathways. Upon cell-extracellular matrix (cell-ECM) contact, PTK2 can be recruited into focal plaques and is rapidly autophosphorylated to recruit other scaffolds and signal molecules to activate the downstream PI3K-AKT signaling pathways . Studies also indicated that HIF-1 gene knockout could effectively inhibit the accumulation of HIF-1 protein and the expression of PTK2 mRNA. Meanwhile, this study also indicated that PTK2 activation was inhibited; the phosphorylation levels of downstream AKT were significantly reduced. These reductions indicated that PTK2 induced the phosphorylation of AKT. The transcriptional activation of PTK2, mediated by HIF-1, protects cells from inactivation and can affect cells' proliferation and differentiation by activating the cell survival signaling pathways and inhibiting the pro-apoptotic signaling pathways.
MET gene is enriched in the PI3K-AKT signaling pathways, located on chromosome 7. Its product is a sort of receptor tyrosine kinase of proteins, with about 110kB in size. MET is mainly expressed in the liver and kidneys, bone marrow cells. The mature MET protein is transmembrane, a dimer complex composed of α and β subunits. There are three structural regions, of which the intracellular domain contains the binding sites for many signal molecules MET activation of the PI3K-Akt signaling pathways can promote cell proliferation and prevent cell apoptosis [38, 39]. In addition to activating the above signaling pathways, MET can also interact with cell death receptors on cell membranes to play an anti-apoptotic role (eg. Fas, FasL). These findings illustrate that MET receptors play a direct role in preventing apoptosis.
RUNDC3B, TDRD1 and NF2 Genes Influence Erythrocytes
RUNDC3B (RUN domain containing 3B) is located on chromosome 7 and is widely expressed in the adrenal glands, brain, liver, small intestines, and other tissues. Although the biological function of RUNDC3B has not been determined, decreased expression of RUNDC3B may result in lymphoid malignancies . In our study, we indicate that the rs527802276 of the RUNDC3B gene was associated with HAPC. Studies have shown that RUNDC3B and RUNDC3A (a Rap2-interacting protein) have high homology, and RUNDC3B also has a similar integration effect like RUNDC3A. The Rap protein family constitutes a subgroup of the Ras superfamily and works as a molecular "switch" regulating various cell functions, such as proliferation, differentiation, and other cell activities . RUNDC3B contains a RUN domain in its N-terminal region, which is an essential component of the mitogen-activated protein kinase (MAPK) cascade. Furthermore, Rap2 interacts with MAP4K4 through its C-terminal citron homology domain. MAP4K4 is a kind of the STE20 protein kinases and regulates c-Jun N-terminal kinase (JNK). Rap2 enhances MAP4K4-induced activation of JNK. So, RUNDC3B appears to play an essential role in activating c-JUN and c-Fos transcription factors, leading to the expression of c-JUN and c-Fos in the nucleus. Its product AP1 can bind to DNA sequences and induce cell proliferation and differentiation.
The TDRD1 gene on chromosome 10 is mainly expressed in the prostate and testes, and a small amount is expressed in the kidneys. Studies have shown that TDRD1 is involved in spermatogenesis. The mutation of the TDRD1 gene is related to spermatogenesis disorder in Han males . In our study, rs11285127 of the TDRD1 gene was involved in the development of HAPC.
NF2 is located on chromosome 22, and its coding product is Merlin. It has a protein with a similar structure to ERM family members. Mutation of the gene is associated with neurofibromatosis type II . In our study, the rs779456792 locus of the NF2 gene was associated with HAPC. The Merlin protein may regulate the PI3K-Akt signaling pathway by interacting with PI3K and other molecules. In this way, the Merlin protein controls cell survival, proliferation, differentiation, and development of RBC, thereby affecting the formation of RBCs .