High altitude can induce extreme pathophysiological changes in travellers that cannot adapt well to the height. Ataltitudes greater than 5500 m the partial pressure of oxygen is critically low which may lead to hypobaric hypoxia in humans. The human body undergoes changes in order to adapt to this environment by increasing anaerobic activities for production of cellular energy, decreasing ATP consumption,erythrocyte expansion , increased haemoglobin count and heart rate etc[1]. These changes require changes in the genetic and epigenetic landscape. These changes are essential for altitude acclimatization. When the human body cannot undergo these adaptations symptoms of acute mountain  sickness,  high  altitude  pulmonary  edema,  and  high  altitude cerebral  edema  (HACE)  can  develop[3].

HACE is a critical and potentially fatal manifestation of mountain sickness whose symptoms include ataxia, fatigue, and altered psychological state. Hypoxia at altitude elicits expression of an array of genes that are responsible for hypoxia-adaptive responses through activation of hypoxia-inducible factor-1 (HIF-1) responsible for angiogenesis (VEG-F) and neuro- hormonal changes (RNS, free radicals) [4]. This results in hypoxia-induced cerebral vasodilation leading leading to intracranial hypertension with elevated capillary pressure and capillary leakage. The disruption of the blood-brain barrier from these stresses leads to subsequent cerebral edema [1].

Recent studies have found that microRNAs play a crucial role in hypoxic adaptation [2]. miRNAs are 17-22 nucleotides long, non- coding, single stranded RNA molecules that regulate both transcriptional and post- transcriptional level adaptations. Due to the critical role they play in cellular functionality, miRNAs have emerged as ideal biomarkers for disease pathogenesis. MiRNAs that are responsive to hypoxic stress are called hypoxiamiRs. HIF is the master sensor of hypoxia and a large number of hypoxiamiRs are induced by HIF stabilization. Regulatory miRNAs are identified by comparison samples from healthy and affected climbers. It has also been found through comparison between natives of high-altitude regions and low-land natives that the same miRNAs are implicated in the ability of the former to survive in such harsh conditions.

Methodology

The first step towards identifying potential miRNA biomarkers would be to mine for data and perform network analysis before moving on to experimental analyses. Protein- protein interaction and miRNA networks have been built using BioGRID with a focus on the genes EPAS1, PPARG, EGLN, Nos3, Apelin1, and ETS1- key regulators of high-altitude adaptations. miRNAs were identified with the help of MicroCosm [3]. Genes, transcription factors, proteins, and miRNAs can be integrated in a network using Cytoscape. In a significant study, altitude responsive network of genes and miRNAs with altered expression levels in HACE was built from data mining from KEGG, STRING, and IntNetDB. Gene- miRNA interaction is proportional to the miRNA expression levels and hence the node weights were defined accordingly. The building of this in siliconetwork enabled identification of important regulatory motifs in hypoxia responsive pathways [2]. Preliminary studies on Tibetan pigs and cattle have identified miRNAs implicated in the hypoxia response. These biomarkers, identified using miRNA seq, regulate MAPK signalling, VEG-F etc[5].

Identification of potential biomarkers relies on comparison between blood samples collected from HACE patients and healthy candidates. RNA isolation followed by miRNA-qPCR profiling is the most commonly used technique. MiRNAs showing significant expressional changes are chosen and then analyzed for pathway enrichment (using softwares like DIANAmirPath). To characterise the genes that these miRNAs target, experiments with HUVEC cells transfected with these miRNAs can be set up to see their up-regulatory/ inhibitory actions[1][2].

HACE
Interactions between hypoxia miRNAs and their target pathways

Conclusion

From the network analysis, it has been found that in patients suffering from HACE, the most commonly altered pathways are the- haemoglobin complex, inflammatory responses, oxidoreductase activity, platelet function, and ribosomal activity [2]. Pathway enrichment studies have identified that EGLN1 and EPAS1 are the most significant regulators of the hypoxia induced factors bot implicated having correlations with haemoglobin levels.

HIF is the master regulatory which directly interacts with miR- 210. MiR-199a, miR- 107, miR-373, miR-23, miR-24 and miR- 26 are induced by HIF family proteins [4]. Using comparative studies of HACE patients and healthy climbers upregulated miRNAs  (miR-629,  miR-1308,miR-124
and miR320c) and down regulated miRNAs (miR-33b, miR- 301a, miR-142- 3p, miR-21 and miR-487b) were identified. MiR-124, miR-320b, miR-340 and miR-142-3 seem to be important regulators because of their higher interactivity [1][2].

Of note, miR124-3p (overexpressed) contributes to (VEGF) signaling, focal adhesion, HIF-1 signaling, transforming growthfactor (TGF)-ß signaling, and Wnt signaling. Decreased expression of miR-16 and miR-20b in HACE disrupts the ion channels and leads to loss of cellular integrity[1][2][4]. HACE is a fatal disease which should be the first to be ruled out in case of mountain sickness. This can be done using identification of miRNA biomarkers from patient blood sample.The various miRNAs mentioned above can be used as potential biomarkers for the same.

References

    [1] Alam,P.,Agarwal,G.,Kumar,R.,Mishra,A.,Saini,N.,Mohammad,G.,&Pasha,
M. Q. (2020). Susceptibility to high-altitude pulmonary edema is associated with circulating miRNA levels under hypobaric hypoxia conditions. American Journal of Physiology-Lung Cellular and Molecular Physiology. doi:10.1152/ajplung.00168.2020
    [2] Chen, F., Zhang, W., Liang, Y., Huang, J., Li, K., Green, C. D., . . . Wang, J. (2012). Transcriptome and Network Changes in Climbers at Extreme Altitudes. PLoS ONE, 7(2). doi:10.1371/journal.pone.0031645
    [3] De,B.,Huajun,X.,Cuihong,Z.,Jun,Z.,Xiaoyan,D.,&Xiaopeng,L.(2013).Systems biology approach to study the high altitude adaptation in tibetans. Brazilian Archives of Biology and Technology, 56(1), 53-60.doi:10.1590/s1516-89132013000100007
    [4] Gupta, A., Sugadev, R., Sharma, Y. K., Ahmad, Y., & Khurana, P. (2018). Role of miRNAs in hypoxia-related disorders. Journal of Biosciences, 43(4), 739-749. doi:10.1007/s12038-018-9789-7
    [5] Kong,Z.,Zhou,C.,Li,B.,Jiao,J.,Chen,L.,Ren,A.,...Tan,Z.(2019).Integrative plasma proteomic and micro RNA analysis of Jersey cattle in response to high-altitude hypoxia. Journal of Dairy Science, 102(5), 4606-4618. doi:10.3168/jds.2018-15515