Systems biology approach to study the high altitude adaptation in tibetans

HUMAN AND ANIMAL HEALTH

Systems biology approach to study the high altitude adaptation in tibetans

Bi De^I; Xiao HuajunI, ^*; Zhou Cuihong^II; Zhou Jun^II; Deng Xiaoyan^II; Liu Xiaopeng^III

^IDepartment of Human Machine and Environment Engineering; Beihang University & Institute of Aviation Medicine; Beijing 100191 - China

^IISchool of Biology Science and Medical Engineering; Beihang University; Beijing 100191 - China

^IIIInstitute of Aviation Medicine; Beijing 100142 - China

ABSTRACT

The aim of this work was to study an integrative systems biology research strategy to construct a network including the protein-protein interactions (PPIs) and microRNAs (miRNAs) and identify the functional biological processes and pathways for high-altitude adaptation in Tibetans. The pathway enrichment analysis revealed that the genes in the network were mainly involved in signling the pathways and the function of microRNAs was concentrated in the signling pathways, which suggested that miRNAs might contribute to the Tibetans high-altitude adaptation through the participation in signaling pathway. These results contribute to better understanding on the high-altitude adaptation of the Tibetans.

Key words: systems biology, Tibetan, high-altitude adaptation, single nucleotide polymorphism, microRNA

INTRODUCTION

The Tibetans live at the Tibetan Plateau, where the average elevations is above 4,000 meters high and every breath of air contains only nearly 60% of the oxygen molecules in the same breath at sea level. The Hypoxia (low oxygen) in the Tibet Plateau is the most severe environmental challenges to the local residents. Most people who live at the sea level will show altitude stress, even acute mountain sickness ranging in severity from headache, pulmonary edema, anorexia, nausea, dizziness or disordered sleep to life-threatening pulmonary or cerebral edema because of a lack of oxygen at high altitudes . However, Tibetans who have been living at very high altitudes for thousands of years have heritable adaptations to extreme altitude; they show genetic adaptation to high altitude . Compared with the people living at the sea level, the feature in the Tibetans for coping with the hypoxic conditions at high altitude is the decreased Hemoglobin (Hb) levels. Yet the complications related with the sustained high Hb levels is seen in non-Tibetan lowlanders when they are exposed to high-altitude conditions . Some genes have shown significant different single nucleotide polymorphisms (SNPs) between the Tibetan and Han populations (Beall et al., 2010; Peng et al., 2011; Simonson et al. 2010; Yi et al. 2010). The Endothelial Per-Arnt-Sim (PAS) domain protein 1 (EPAS1; also termed HIF2α) and EGL-nine homolog-1 (EGLN1) play key role in high-altitude adaptation of the Tibetans. Both the EPAS1 and EGLN1 are involved in the hypoxic pathways as key regulator during the chronic hypoxia. The EPAS1 and EGLN1, which have undergone positive selection, are associated with the Hb levels in the Tibetan populations. The SNPs at EPAS1 and EGLN1 provides evidence of a genetic contribution to high-altitude adaptation of the Tibetan populations.

The EPAS1 is the key regulator during the chronic hypoxia, and it directly regulates the genes such as vascular endothelial growth factor (VEGF), erythropoietin (EPO), and endothelial nitric oxide synthase (eNOS) to cope with the hypoxic conditions. The EGLN1 is a negative regulator of the EPAS1 and it targets the EPAS1 and Hypoxia inducible factor 1 α (HIF1A) for degradation under the normoxic conditions. Under the hypoxic conditions, the degradation effect of the EGLN1 is weakened so that the increase of the EPAS1 protein could initiate the expression of the downstream genes, including VEGF, EPO and eNOS whose product induces red blood cell (RBC) production. The HIF1A is a key regulator of the tissue response to the hypoxia, The EGLN1 negatively regulates the activity of the HIF1A . The EPAS1 and EGLN1, as well as other proteins, specifically the HIF1A that have interactions with them working together, play a key role in adaptation to high altitude of the Tibetans.

Although it has been reported that the PPARA has also undergone positive selection and are associated with the Hb levels in the Tibetan populations and that the haplotype variation at the PPARA contribute to high-altitude adaptation of the Tibetan populations (Simonson et al., 2010), other studies have shown that the tag SNPs at PPARA has no significant allele frequency divergence between the Tibetan and non-Tibetan populations . However, some genes tag SNPs have shown significant frequency differences between the Tibetan and non-Tibetan populations . This shows that more in-depth studies of these genes function in high-altitude adaptation of the Tibetans are needed. Thus, this work focussed on studying the EPAS1 and EGLN1 playing key role in high-altitude adaptation of theTibetans. The microRNAs (miRNAs), the ～22 nucleotide (nt) small RNAs involved in the control of gene expression , are important regulators of eukaryotic gene expression and the SNP at miRNAs target genes are associated with the human disease . As the miRNAs target genes, the EPAS1 and EGLN1 have significant SNPs between the Tibetan and Han populations; hence, studying the function of miRNAs could provide better understanding on Tibetans high-altitude adaptation. Furthermore, the protein-protein interactions (PPIs) could provide insight into protein function and facilitate the modeling of functional pathways to elucidate the molecular mechanisms of biological processes. Owing to the importance of the PPIs involved in the EPAS1, EGLN1 and HIF1A, as well as the miRNAs as the key post-transcriptional regulators of them, the PPIs and miRNAs network constructe]ion is required through the database mining to get better understanding of the role of the EPAS1 and EGLN1 in high-altitude adaptation process. What biological processes and biological pathways are involved in the PPIs network and miRNAs network, and whether the miRNAs contribute to the high altitude adaptation are still relatively unexplored. For this reason, this work further performed the systems biology study to explore the functional biological processes and pathways involved in the hypoxia adaptation, which could provide better insight to the high-altitude adaptation process. Furthermore, the systems biology approach could improve the understanding of the molecular basis of high-altitude adaptation and might identify the key functional pathways or relate molecular events of the disease caused by the high-altitude hypoxia.

METHODS

PPIs and miRNAs network

The PPI databases such as BioGRID (http://thebiogrid.org/) and Human Protein Reference Database (HPRD) (http://www.hprd.org/) were used to obtain the PPIs, including the EPAS1, EGLN1 and HIF1A. Although more than a hundred genes are regulated by the HIF1A, EPAS1 and EGLN1are the two key HIF regulatory genes encoding the transcription factors that induce the downstream genes when cellular oxygen levels decrease. Previous studies have shown that the incompleteness of the gene or proteins annotation repositories does not necessarily impair the computational function prediction . In order to focus on the biological function enrichment analysis of the high-altitude adaptation of Tibetans, the database were mined and first-level PPIs of EPAS1, EGLN1 and HIF1A were constructed, which played key role in high-altitude adaptation of the Tibetans. The PPIs network included the indirect second-level PPIs.

MiRNAs, the regulators of the EPAS1, EGLN1 and HIF1A, were obtained from the MicroCosm Targets. The PPIs and miRNAs were integrated to one network by the Cytoscape version 2.8.1, an open source software platform based on JAVA for visualizing and analyzing the genetic and molecular interaction complex networks and integrating these such as protein-protein, miRNA-gene, and genetic interactions. The PPIs and miRNAs were loaded into Cytoscape with different size and color node for visualizing as networks.

Functional enrichment analysis of genes in PPIs network

In order to assess the relative functional enrichment of the genes corresponding to the proteins in the PPIs, the Gene Ontology (GO) Consortium, Genecodis vision 2.0 online tool, a web server application for functional analysis of in the large lists of genes or proteins that integrated different sources of information and found the modular patterns of interrelated annotations, was used for functional cluster analysis, including the biological processes, molecular function and biological pathways. The biological processes annotations were obtained from the Gene Ontology (http://www.geneontology.org/) and pathway annotations were obtained from the KEGG. All the data base es selected the Homo sapiens as reference.

Functional enrichment analysis of miRNAs in network

In order to assess the relative functional enrichment of miRNAs corresponding to the genes in the network, the miTALOS (http://mips.helmholtz-muenchen.de/mitalos/index.jsp) online tool a web server application for providing the insight into miRNA-mediated regulation of signaling pathways, was used for the biological pathways cluster analysis. The Target, ScanTarget, SpyRNA22 and PicTar programs, which were compiled in miTALOS, were used together for the target gene prediction in order to get more biological pathways. The biological pathways annotations were obtained from the KEGG, which were compiled and stored in the miTALOS. The organism was human; the enrichment was 1.00, and P-Value (E) was 0.05.

RESULTS AND DISCUSSION

A network containing 92 proteins and 46 miRNAs (nodes) and 157 interactions (edges) was constructed (Fig 1).

Genes functional enrichment analysis

To find the functional biological processes and pathways, which contributed to the high-altitude adaptation, each gene in the network was categorized based on the biological processes (Table 1), cellular component (Table 2), molecular function (Table 3), and pathways (Table 4).

The biological processes enrichment results revealed that the genes in the PPIs involved in response to the hypoxia, positive and negative regulation of transcription from the RNA polymerase II promoter, regulation of transcription, chromatin remodeling and negative regulation of apoptosis, in addition, nerve growth factor receptor signaling pathway and negative regulation of myotube differentiation were included (Table 1). The molecular function enrichment analysis showed that the genes located in the nucleus, nucleoplasm and cytoplasm (Table 2) participated in the transcription factor binding, protein binding, DNA binding, NAD-dependent histone deacetylase activity and ubiquitin binding (Table 3). The pathways annotation results revealed the ErbB signaling pathway, Wnt signaling pathway, MAPK signaling pathway and cancer pathway (Table 4).

MiRNAs functional enrichment analysis

To further investigate the relationship between the miRNAs and molecular pathways, which were in the major human organs, including cerebellum, heart, liver, kidney, lung and smoothmuscle, the miRNAs involved in the biological pathways, enrichment analysis was explored by the miTALOS online tool. The pathways annotation analyses showed that the miRNAs were involved in the MAPK signaling pathway, T cell receptor signaling pathway and insulin signaling pathway (Table 5).

These results together reflected the complicated biological processes and pathways of the EPAS1 and EGLN1 involved, which were intimately associated with the high-altitude adaptation of the Tibetan populations. The system biology approach provided further understanding of the function of miRNAs that were regulators of the EPAS1, EGLN and HIF1A. Interestingly, the miRNAs functional cluster results showed that such function as MAPK signaling pathway was consistent with the genes biological pathway cluster analysis results. The pathways enrichment of the miRNAs analysis indicated that the miRNAs might contribute to the Tibetans high-altitude adaptation through participating in signaling pathway.

The EGLN1 is involved in cellular oxygen sensing and certain EGLN1 mutations lead to polycythemia. The higher expression of EGLN1 is inversely correlated to HIF activity. The EGLN1 is associated with lower hemoglobin levels in the Tibetans. There is strong and significant association between the hemoglobin concentration and haplotype variation at the EGLN1. Yet, high hemoglobin acclimatization can cause stokes, heart attacks and pulmonary embolisms.

The present results showed that most proteins in the PPIs network were involved in the hypoxia response mainly participate in signaling pathways. The EPAS1 and EGLN1 were the key genes that provided the Tibetans the hypoxia adaption ability. The hypoxia-response pathways in the humans are very complex. The biological processes, molecular function and pathways involved in the EGLN1 and EPAS1 help to understand the high-altitude adaptation of Tibetans.

Tibetans have the hypoxia adaptation ability and hypoxic preconditioning can reduce the severity of induced or inherited degeneration in the tissues such as the brain, heart and the retina. The EPAS1 is a transcription factor regulating the gene expression in response to the hypoxia, and the expression of EPAS1 is cell type-restricted. It is predominantly expressed in the vascular endothelial cells, lung epithelial cells and cardiac myocytes. In addition to the hypoxic response function, the EGLN1 is being considered as an important pharmacological target. Thus, it could be hypothesized that whether the genes, such as EPAS1 and EGLN1 with significant difference SNPs that provide the Tibetans high-altitude adaptation ability, could make them avoid some kind of disease related with the hypoxia would be very interesting.

In conclusion, the system biology analysis of the EPAS1 and EGLN1, which have significant difference SNPs between the Tibetan and Han populations contribute to provide better understanding on the Tibetans high-altitude adaptation.

ACKNOWLEDGEMENT

This work was supported by the grants from the National Natural Science Foundation of China (11072023 to X. D.).

Received: August 01, 2011

Revised: March 06, 2012

Accepted: September 21, 2012

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Publication Dates

• Publication in this collection
  09 Apr 2013
• Date of issue
  Feb 2013

History

• Received
  01 Aug 2011
• Accepted
  21 Sept 2012
• Reviewed
  06 Mar 2012