Study Of The Transcriptional Signatures And Its Pathophysiology Significance During High Altitude Acclimatization And Un-acclimatization

Background:High altitude is a special area around the world with several basic features,including low barometric pressure,hypoxia,cold,dehydration,and solar radiation.Among them,hypoxia is the main factor which lead to undesirable effects on human live in high altitude because the other factors can be avoided by proper clothing or shelter.Altitude acclimatization is the physiological process comprises a number of responses by different system in the body,which takes place in the body on exposure to hypoxia at altitude.Many studies have revealed that changes involved in acclimatization occur in various systems and with varying time courses.Many people can maintain homeostasis needed for body function through acclimatization though at a lower partial pressure.However,acute and chronic high altitude illness will occur in people who exposed in high altitude with a poor acclimatization.Acute high altitude illness includes acute mountain sickness(AMS),high altitude pulmonary edema,and high altitude cerebral edema.Among them,AMS is the most common acute high altitude illness,and also is the major threaten for people who ascend to high altitude acutely;the median of AMS incidences without prophylaxis was 60% in randomized trails.AMS can progress to high altitude cerebral edema in severe cases,which is life-threatening.Chronic high altitude disease is comprised of high altitude polycythemia(HAPC)and high altitude pulmonary hypertension.HAPC is the most common chronic high altitude disease and characterized as excessive erythropoiesis and significantly increased hemoglobin concentration,which will lead to injuries in multiple organs and multi-systems.HAPC is the major threaten for people who exposed to high altitude for a long time.When people returned to plain after living in the high altitude for a period of time,the changes of body in high altitude acclimatization will reprogram for plain.The process was defined as high altitude de-acclimatization.During the process,some people present clinical symptoms,such as sleepiness,deterioration of the memory and cognition.The study of molecular underpinnings for high altitude acclimatization and un-acclimatization is the core project of high altitude medicine.Although many studies have demonstrated the physiological and biochemical changes in high altitude acclimatization,the molecular mechanism of high altitude acclimatization remains unknown.The previous studies of molecular mechanisms for AMS and HAPC have revealed that AMS and HAPC are complex diseases caused by multiple genes and the interaction of genes and high altitude hypoxia.Thus,it is essential to study the molecular mechanisms of AMS and HAPC at system level.Gene expression is a major way in which cells would maintain homeostasis,and change state.Unlike the nuclear genome,whose composition and size are essentially static,the transcriptome often changes.Understanding the transcriptome is essential for revealing molecular constituents of cells and tissues under a specific development stage or physiological condition,and also for understanding disease.Therefore,the study here explored the transcriptional signatures of Han Chinese population during high altitude acclimatization and de-acclimatization with WGCNA algorithm based on RNA-Seq data,and illustrated its pathophysiology significance with gene annotation database and prior knowledge to illustrate the molecular underpinnings of high altitude acclimatization.Moreover,we compared the changes of gene expression between AMS/HAPC patients and healthy individuals using network analysis to find the gene co-expression pattern and hub genes associated with AMS and HAPC.The results of the study will be helpful to make a comprehensive understanding for high altitude acclimatization and provide a new avenue of intervention target for AMS and HAPC.Methods:1.We enrolled two independent healthy male volunteers of Han Chinese in the study.(1)We dynamically recorded the blood pressure,heart rate and peripheral capillary oxygen saturation of volunteers during high altitude acclimatization and de-acclimatization process at 6 time points,including pre-exposure to high altitude,the early phase of high altitude(5300m)exposure(3d),the middle phase of high altitude exposure(4m),the later stage of high altitude exposure(1y),the early phase of return to plain(1m),and the later stage of return to plain(6m).During the process,blood samples were collected for RNA-Seq,hemoglobin and cytokine concentration detection.(2)Blood samples from volunteers who acutely ascend to high altitude(3648m)were collected for measuring circulating microRNAs expression before high altitude exposure.The clinical symptoms were recorded during high altitude exposure.2.The total RNA was extracted form blood samples,and PE sequencing library was constructed according to the manufacture's instruction.Then RNA-Seq was performed with Illumina Hiseq 2000 platform.3.After data filtering and quality evaluation,we matched the paired-end reads to the human reference genome(hg19).The gene expression was calculated RSEM package.Differentially expressed genes were screened by edgeR package of R software,and the |Fold change| ? 2 and FDR ? 0.05 were chosen as cut-off criterion.WGCNA algorithm was used to acquire the transcriptional signatures of different stages of high altitude acclimatization and de-acclimatization.The biology process of transcriptional signatures was identified using clusterProfiler and REVIGO software based on Gene Ontology(GO)database.Gene co-expression network was constructed based on the relatedness among genes to find out the hub genes in each module.4.RPKM was used to quantify the transcript expression of AMS and non-AMS individuals.Differentially expressed transcripts were identified by comparison of post-exposure and pre-exposure expression values in each group using density based pruning algorithm,respectively.We used the GO tool DAVID to determine enriched GO terms in the differentially expressed gene set.Then,the Gene Functional Classification tool from the DAVID suite was used to cluster enriched GO terms(biological processes)with the classification stringency set to medium and all other options set to default.The topological overlap matrix was calculated in AMS and non-AMS individuals,then,comparison between the two groups was performed.5.The plasma concentration of IL10,CCL8 and IL17 F in AMS and non-AMS were measured using ELISA.Moreover,the concentration of IL10 was confirmed with chemiluminescent immunometric assay using an IMMULITE? 1000 autoanalyzer with commercially available kits which is a method of diagnostic use in the study of inflammatory disease.6.Circulating microRNAs expression was measured by miRCURYTIM LNA Array and quantitative reverse-transcription polymerase chain reaction.The prediction efficiency of circulating microRNAs in AMS susceptibility was evaluated by OptimalCutpoints package.7.To explore the potential biological relevance of microRNAs signature,we predicted target genes of microRNAs using microTCDS and TarBase.GO enrichment analysis was performed for target genes using DIANA-miRPath which deciphers microRNA function with experimental support.8.The gene expression of HAPC and non-HAPC was calculated RSEM package.Differentially expressed genes were screened by edgeR package of R software,and the |Fold change| ? 2 and FDR ? 0.05 were chosen as cut-off criterion.The biology process of differentially expressed genes was identified using clusterProfiler and REVIGO software based on GO database.Spearman correlation coefficient was used to calculate the correlations among genes in HAPC and non-HAPC groups,respectively.Concentric software was employed to cluster the genes in the gene co-expression network based on the degree of genes on k0 and k1 levels.Results:1.In the early phase of high altitude exposure,genes involved in dopamine metabolic process,ion transport and hemoglobin biosynthetic process specifically expressed.Among them,MXI1,RNF10,TRIM58,GLRX5 and BPGM were the hub genes.In the middle phase of high altitudeexposure,the expression pattern of genes was similar to that of pre-exposure to high altitude.In the later stage of high altitude exposure,genes associated with phosphorus metabolic process,regulation of immune system process and MAPK cascade,endocytosis and vesicle-mediated transport specifically expressed.Among them,SMARCD2,CDK9 and RIC8 A were the hub genes.2.After return to plain,genes involved in macromolecule metabolic process,regulation of cell division and cell cycle specifically expressed in the early phase.Among them,MTF2,ZFR,CAND1,DEPDC1,DEPDC1 B,CCNA2,CDC6,CDC20,CCNB2,CCNB1 and ANLN were the hub genes.In the later stage,genes involved in immune system process,innate immune response and cytokine-mediated signaling pathway specifically expressed.Among them,ZBP1 and STAT2,IFIT1,IFIT2 and IFIT3 were the hub genes.3.Immune and inflammatory responses were overrepresented in participants with AMS,but not in non-AMS individuals(enrichment score: 3.44,3.27,respectively).Antiinflammatory cytokine IL10 and inflammation cytokines IF17 F and CCL8 exhibited significantly different genetic connectivity in AMS compared to that of non-AMS individuals based on network analysis.IL10 was down-regulated and both IF17 F and CCL8 were un-regulated in AMS individuals.Moreover,the serum concentration of IL10 significantly decreased in AMS patients after exposure to high altitude(p = 0.001)in another population(n=22).There was a large negative correlation between the changes of IL10 concentration,r(22)=-0.52,p = 0.013,and Lake Louise Score.4.Brfore high altitude exposure,the expressions of miR-369-3p,miR-449b-3p,and mi R-136-3p in AMS patients were significant higher compared to that in non-AMS individuals.The combination of mi R-369-3p,miR-449b-3p,and miR-136-3p could efficiently discriminate AMS from Non-AMS(area under the curve 0.986,95%CI 0.970–1.000,p < 0.001,LR+: 14.21,LR–: 0.08).This signature yielded a 92.68% sensitivity and 93.48% specificity for AMS vs.Non-AMS.Cellular nitrogen compound metabolic process and neurotrophin TRK receptor signaling pathway,were over-represented in target genes of the microRNA signature.5.Compared to non-HAPC individuals,there are 311 up-regulated genes in HAPC individuals significantly enriched in cell proliferation and regulation of immune system process.The average degree of up-regulated gene co-expression network in HAPC is 22.64,it is 2 times than that in non-HAPC(11.85).IFT20 and MRPL22 have higher betweeness centralities in gene co-expression network of HAPC(0.50 and 0.46,respectively).ITGAV and TPRKB have higher betweeness centralities in gene co-expression network of non-HAPC(0.50 and 0.37,respectively).Conclusions:1.In the early phase of exposure to high altitude,MXI1,RNF100,TRIM58 and BPGM regulated genes involved in dopamine metabolic process,ion transport and hemoglobin biosynthetic process to enhance oxygen intake,transport and the oxygen release ratio of oxyhaemoglobin for acclimated to high altitude.With a prolonged stay at high altitude,SMARCD2,CDK9 and RIC8 A regulated genes associated with phosphorus metabolic process,regulation of immune system process and MAPK cascade,endocytosis and vesicle-mediated transport to promote erythrocyte proliferation,angiogenesis,and oxygen utilization of tissue cells.Erythrocyte proliferation could enhance the oxygen transport.Promoting angiogenesis could shorten the oxygen dispersion distance.All these changes will help people to acclimate to high altitude well.Activation of immune system maybe will protect body from hypoxia injury and maintain the body homeostasis though at a lower partial pressure.2.After return to plain,MTF2,DEPDC1,DEPDC1 B,CCNA2,CDC6,CDC20,CCNB2,CCNB1 and ANLN regulated genes involved in macromolecule metabolic process,regulation of cell division and cell cycle to promote the proliferation of cells in the early phase for maintaining the body homeostasis.With a prolonged stay at plain,ZBP1,STAT2,IFIT1,IFIT2 and IFIT3 regulated genes involved in immune system process,innate immune response and cytokine-mediated signaling pathway to remove endogenous damage factor and repair tissue.3.Immune and inflammatory responses play a pivotal role in the pathogenesis of AMS.The reduction of IL10 and increment of CCL8 and IL17 F after high altitude exposure caused by changed gene co-expression pattern in AMS patients maybe the key point in inflammatory response in AMS.4.The study here,for the first time,describes a signature of three circulating microRNAs as a robust biomarker to predict the susceptibility of AMS before exposure to high altitude.5.The over-regulation of genes involved in cell proliferation and immune response and its specific co-expression pattern are closely related to the occurrence of HAPC.IFT20 and MRPL22 are the key genes in the process,suggesting that they may be important targets for HAPC treatment.In conclusion,we measured the whole blood transcoptome of Han Chinese during high altitude acclimatization and de-acclimatization process,dynamically.We illustrated the transcriptional signatures and its pathophysiology significance at the different stages of high altitude acclimatization and de-acclimatization process to promote the comprehensive understanding of high altitude acclimatization.We compared the changes of gene expression between AMS/HAPC patients and healthy individuals using network analysis to find the gene co-expression pattern and hub genes associated with AMS and HAPC.These results provide a new avenue of intervention target for AMS and HAPC.And the study here,for the first time,describes a signature of three circulating microRNAs as a robust biomarker to predict the susceptibility of AMS before exposure to high altitude.