| Background:China is the country with the largest high-altitude area and the largest resident population around the world.Nowadays,about 60 million residents live in the high-altitude area Recently,with the development of social economy and national defense at high altitude,more than 10 million people ascend to the high-altitude area every year,for tourism,commerce,construction and military guarding.The natural environment of high-altitude area is very harsh,hypobaric hypoxia can cause acute mountain sickness(AMS)and high-altitude polycythemia(HAPC),which seriously affect the body health.AMS is a prevalent disease among people exposed to high altitudes of>2,500 m and presents as a combination of several symptoms,such as headache,dizziness,gastrointestinal symptoms,and fatigue.The incidence of AMS is pretty high,varying from 30%to 90%.Moreover,AMS can significantly reduce the operational ability of people acutely exposed to high altitudes,thus disturbing their daily work.More seriously,the severe form of AMS can even progress to high altitude cerebral edema(HACE),which have life-threatening consequences.Indeed,AMS has become a crucial public health problem owing to a significant rise in the number of people ascending per year.HAPC is a prevalent chronic mountain sickness,characterized by excessive erythropoiesis.The incidence of HAPC varies from 1.21%to 24.0%among residents at 3,000-4,500 m.HAPC seriously affect body health,the excessive erythropoiesis could lead to blood viscosity,microcirculation dysfunction,and severe hypoxemia.More seriously,the severe form of HAPC may cause thromboembolism in various organs,thus resulting in sudden death.Therefore,HAPC has become the major health threaten for the residents at high altitude.Currently,there are no drugs with high specificity and low side effects for the treatment of AMS and HAPC;thus,the early disease prevention is very important.However,the biomarkers for predicting the risk of AMS and HAPC are still lacking.The main reason for this awkward condition is that,the molecular biological mechanisms of AMS and HAPC are not clear yet.Therefore,there are urgent needs for making intensive research on the pathogenesis of AMS and HAPC from a new perspective,to find effective treatment targets and biomarkers for risk predictionMicroRNAs are 18?24-nucleotide long,single-stranded,non-coding RNAs,which are important class of gene-modulators for various physiological and disease processes,such as cell cycle,growth,development,differentiation,apoptosis,and inflammatory response Moreover,hypoxia can alter the expression of various microRNAs,thus involved in physiological processes for hypoxia compensation and hypoxia-induced pathophysiological processes.It has been found that abnormal microRNA expression is closely related to the occurrence of multiple hypoxia-associated diseases,such as coronary heart disease,stroke,hepatic and renal ischemia injury,acute respiratory distress syndrome,etc.These studies not only provide a new way to elucidate the pathogenesis of the disease,but also find effective prevention and treatment targets.However,whether microRNAs involve in the pathogenesis of AMS and can be used as a biomarker to predict the risk of AMS,are still not clear.At the same time,microRNAs are also involved in the proliferation,differentiation and maturation of erythroid cells,and play an important role in the pathogenesis of various blood diseases,such as polycythemia veranda,secondary polycythemia and bone marrow proliferative tumors However,the roles of microRNAs in HAPC pathogenesis are still not fully understoodTherefore,this study conducted the following five parts experiments,to explore the microRNA characteristics of AMS and HAPC,and to figure out their roles in the pathogenesis of diseases.In the first part,microRNA expression profile array was used to detect the plasma samples of AMS patients and Non-AMS participants after high-altitude exposure,to reveal the microRNA expression profile characteristics of AMS patients.Furthermore,Gene Oncology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment were conducted for analysis target genes of AMS related microRNAs,to explore the molecular biological processes and signal pathways regulated by these microRNAs,and to provide new sights for AMS pathogenesis.In the second part,we choose the miR-181b-5p,which was up-regulated in AMS patients,as the target for experiments of molecular biology.Then,we conducted bioinformatics analysis and experiments of molecular biology in vivo and vitro to clarify its role in the inflammatory pathogenesis of AMS.In the third part,qRT-PCR was used to detect the microRNA expression level of plasma and saliva samples of AMS patients and Non-AMS participants at sea level(before acute high-altitude exposure),to find efficient biomarkers for AMS risk prediction.In the fourth part,RNA-seq was applied to detect the plasma samples of HAPC patients and healthy controls at the same altitude,to reveal the microRNA expression profile characteristics of HAPC patients.Furthermore,GO and KEGG enrichment were used for analysis the target genes of HAPC related microRNAs,to explore the molecular biological processes and signal pathways regulated by these microRNAs,and to provide new sights for HAPC pathogenesis.In the fifth part,we choose the miR-144-3p,which was up-regulated in HAPC patients,as the target for experiments of molecular biology Then,we conducted bioinformatics analysis and experiments of molecular biology to clarify its role in the oxidative stress injury of erythroid cells and its association with the pathogenesis of HAPCMethods:1.Detection the microRNA expression profiles of AMS patients(1)The study subjects were AMS patients(AMS)and those without AMS(Non-AMS).AMS diagnosis was performed according to the Lake Louis Score System(LLS)(2)Collect the demographic data,physiological indicators and plasma samples of the study subjects.(3)Perform the microRNA expression profile array to detect the microRNA(4)Perform bioinformatics analysis for AMS related microRNAs' target genes(5)Verify the important differential microRNAs screened by profile array via qRT-PCR2.The study on biological functions of miR-181b-5p(1)Perform bioinformatics analysis for miR-181b-5p's target genes to identify the signaling pathways regulated by it.Then,select the miR-181b-5p'target gene based on associated signaling pathways(2)Eight-week-old male C57BL/6 mice were selected as animal model and divided into two groups:(?)control group,mice were normally fed for 24 h at sea level;(?)high altitude cerebral edema(HACE)group,mice were injected with lipopolysaccharide(LPS)(dose:0.5 mg/kg)via tail vein,then,fed in hypobaric hypoxia chamber(simulated altitude:6,000 m)for 24h.(3)Perform qRT-PCR to detect the expression levels of miR-181b-5p,IL-1?,IL-6 and TNF-? in blood white blood cells of animal model(4)RAW264.7 cell line was selected as the cell model.RAW264.7 cells were divided into two groups:(?)control group,cell was cultured in normal medium and 21%O2 for 24 h;(?)the hypoxia-LPS combined stimulation group,cell was cultured in LPS contained medium(100 ng/mL)and 1%O2 for 24 h(5)RAW264.7 cells were transfected with miR-181b-5p mimic,inhibitor and negative control.Then,perform qRT-PCR and enzyme linked immunosorbent assay(ELISA)to detect the IL-1?,IL-6 and TNF-? mRNA levels in cell,and IL-1?,IL-6 and TNF-? concentrations in cell supernatant,respectively(6)"Protein kinase C delta(PRKCD)" was confirmed as the target gene of miR-181b-5p by double luciferase reporter assay3.Verification the role of plasma and saliva microRNA as biomarkers for AMS risk prediction(1)The study subjects were AMS patients(AMS)and those participants without AMS(Non-AMS).(2)Collect the demographic data,physiological indicators and plasma samples of the study subjects.(3)Plasma and saliva samples of AMS and Non-AMS groups at sea level were collected(4)Plasma samples of AMS and Non-AMS groups at high altitude were collected(5)Plasma miR-1183,miR-3654,miR-15b-5p and miR-23b-5p expression levels at sea level,were detected by qRT-PCR.(6)Plasma IL-1?,IL-6 and TNF-? concentrations at high altitude,were measured by ELISA.(7)Salivary miR-134-3p and miR-15b-5p expression levels at sea level,were evaluated by qRT-PCR.(8)Perform bioinformatics analysis for target genes of miR-1183,miR-15b-5p,miR-23b-5p and miR-134-3p to identify the biological processes regulated by these microRNAs.4.Detection the microRNA expression profiles of HAPC patients(1)The study subjects were HAPC patients and those without HAPC at the same altitude The HAPC diagnostic criteria were based on the consensus statement by the International Society for Mountain Medicine(ISMM)at the 6th World Congress on Mountain Medicine and High-Altitude Physiology(2)Collect the demographic data,physiological indicators and plasma samples of the study subjects.(3)Perform the RNA-seq to detect the microRNA(4)Perform bioinformatics analysis for HAPC related microRNAs' target genes(5)Verify the important differential microRNAs screened by RNA-seq using qRT-PCR5.The study on biological functions of miR-144-3p(1)Perform bioinformatics analysis for miR-144-3p's target genes to identify the signaling pathways regulated by this microRNA.Then,select the miR-144-3p'target gene based on associated signaling pathways(2)Perform qRT-PCR to detect the expression levels of miR-144-3p in blood erythrocytes of HAPC patients and healthy controls at the same altitude(3)Detect the concentration of reactive oxygen species(ROS),malonaldehyde(MDA),superoxide dismutase(SOD)and glutathione(GSH)in blood erythrocytes of HAPC patients and healthy subjects at the same altitude via biochemical array(4)Six-week-old male SD rats were selected as animal model and divided into two groups:(?)control group,rats were normally fed for 28 d at sea level;(?)HAPC group,rats were fed in hypobaric hypoxia chamber(simulated altitude:5,800 m)for 28 d(5)Perform qRT-PCR to detect the expression levels of miR-144-3p in blood erythrocytes of rat HAPC group and control group(6)Detect the concentration of reactive oxygen species(ROS),malonaldehyde(MDA),superoxide dismutase(SOD)and glutathione(GSH)in blood erythrocytes of rat HAPC group and control group via biochemical array(7)K562 cell line was selected as the cell model.K562 cells were divided into two groups:(?)control group,cell was cultured in normal medium and 21%O2 for 6 h;(?)the oxidative injury group,cell was cultured in hydrogen peroxide contained medium(10 uM/mL)and 21%O2 for 6 h(8)K562 cells were transfected with miR-144-3p mimic,inhibitor and negative control Perform qRT-PCR to detect the SOD1,GCLC,GCLM,CAT,GPX1 and NQO1 mRNA levels in cell.Then,detect the ROS MDA concentrations via biochemical array.(9)"Nuclear factor E2-related factor 2(NRF2)" was confirmed as the target gene of miR-144-3p by double luciferase reporter method.Results:1.MicroRNA expression profile characteristics of AMS(1)MicroRNA expression profiles of AMS patients and those without AMS were significantly differentThere were 93 microRNAs were significantly different in AMS patients and those without AMS.Among them,56 microRNAs were significantly up-regulated and 37 were significantly down-regulated in AMS patients(all,Foldchange?2,p<0.05).(2)AMS related microRNAs were mainly involved in regulating signal pathways associated with hypoxia adaptation,energy metabolism,angiogenesis and inflammatory responseThe functions of AMS related microRNAs were mainly involved in regulating signaling pathways,such as hypoxia adaptation(HIF-1 signaling pathway),energy metabolism(cAMP signaling pathway),angiogenesis(VEGF signaling pathway),and inflammatory response(MAPK signaling pathway,NF-?B signaling pathway,toll-like receptor signaling pathway,NOD-like receptor signaling pathway,and TNF signaling pathway).(3)The results of microRNA array were highly repeatableThe results of qRT-PCR showed that,compared with those without AMS,the plasma expression levels of miR-676-3p,miR-181b-5p,miR-193b-5p and miR-3591-3p in AMS patients were significantly up-regulated,which were consistent with the results of microRNA array(all,p<0.01).(4)miR-676-3p,miR-181b-5p and miR-3591-3p were identified as diagnostic biomarkers for AMSROC curve results showed that the diagnostic power of miR-676-3p,miR-181b-5p and miR-3591-3p for AMS was 0.713(95%CI=0.588-0.838,p<0.01),0.735(95%CI=0.614-0.855,p<0.001)and 0.805(95%CI=0.700-0.911,p<0.001),respectively.2.miR-181b-5p served as a negative regulator of inflammatory response(1)miR-181b-5p was involved in inflammatory response pathwaysBioinformatics analysis showed that the miR-181b-5p was involved in signaling pathways related to inflammatory response,such as MAPK signaling pathway,NF-?B signaling pathway,toll-like receptor signaling pathway,and NOD-like receptor signaling pathway.Among them,PRKCD,an important gene in the NF-?B signaling pathway,was identified as the target gene of miR-181b-5p(2)miR-181b-5p was significantly upregulated in HACE modelCompared with the control group,miR-181b-5p,IL-1?,IL-6 and TNF-? were significantly up-regulated in white blood cells of mice with HACE(all,p<0.05).These results suggest the relationship between miR-181b-5p and inflammatory response in vivo(3)miR-181b-5p might reduce macrophage inflammatory response by inhibiting PRKCDThe overexpression of miR-181b-5p in RAW264.7 cell,could significantly down-regulate the expression levels of IL-1?,IL-6 and TNF-? mRNA in cell,and decrease the concentrations of IL-1?,IL-6 and TNF-? in the cell supernatant(all,p<0.01).The suppressing expression of miR-181b-5p in RAW264.7 cells,could up-regulate the expression levels of IL-1?,IL-6 and TNF-? mRNA in cell and increase the concentrations of IL-1?,IL-6 and TNF-? in the cell supernatant(all,p<0.01).Double luciferase reporter assay confirmed PRKCD as the target gene of miR-181b-5p(p<0.01)3.Plasma and salivary microRNAs at sea level could be used as biomarkers for AMS risk prediction(1)Plasma microRNAs at sea level could be used as biomarkers for AMS risk predictionThe combination of plasma miR-1183,miR-15b-5p and miR-23b-5p could efficiently discriminate Non-AMS from AMS at sea level(AUC=0.872,95%CI=0.836-0.903,p<0.001).(2)The combination of miR-1183,miR-15b-5p and miR-23b-5p associated with inflammatory responseResults of GO analysis showed that miR-1183,miR-15b-5p and miR-23b-5p were involved in the regulation of the immune system process,innate immune response,MAPK signaling pathway,MyD88-Toll-like receptor signaling and other classical inflammatory response pathways.Correlation analysis showed that miR-1183,miR-15b-5p and miR-23b-5p were significantly correlated with plasma inflammatory factors(IL-1?,IL-6 and TNF-?)(all,p<0.001).(3)Salivary microRNAs at sea level could be used as non-invasive biomarkers for AMS risk predictionThe combination of salivary miR-134-3p and miR-15b-5p could efficiently discriminate Non-AMS from AMS at sea level(AUC=0.811,95%CI=0.731-0.876,p<0.001)(4)miR-134-3p and miR-15b-5p involved in inflammatory responseResults of GO analysis showed that miR-134-3p and miR-15b-5p could jointly regulate classic inflammatory response pathways such as MAPK and Toll-like receptor signaling pathways.4.MicroRNA expression characteristics of HAPC patients(1)MicroRNA expression profiles of HAPC patients and healthy controls were significantly differentThe RNA-seq results showed that there were 44 microRNAs significantly different in HAPC patients and healthy controls at the same altitude.Among them,33 microRNAs were significantly up-regulated and 11 were significantly down-regulated in HAPC patients(all,Foldchange?2,p<0.05)(2)HAPC related microRNAs involved in the regulation of multiple signaling pathwaysThe above HAPC-related microRNA involved in the regulation of multiple signaling pathways,such as sphingolipids signaling pathway,endocytosis signaling pathway,mitochondrial autophagy signaling pathway,Ras signaling pathway,TNF signaling pathway,MAPK signaling pathway and Hedgehog signaling pathway(3)The results of RNA-seq were highly repeatableThe results of qRT-PCR showed that,compared with healthy controls at the same altitude,the plasma expression levels of miR-144-3p,miR-210-3p,miR-19b-3p and miR-21-3p in HAPC patients were significantly up-regulated,which were consistent with the trend of RNA-seq results(all,p<0.01)5.miR-144-3p served as a negative regulator of antioxidant injury pathway(1)miR-144-3p might be involved in antioxidant injury pathwaysBioinformatics analysis showed that the miR-144-3p was involved in HGF receptor signaling pathway,NRF2-antioxidant injury signaling pathway,Sonic Hedgehog signaling pathway and MAPK signaling pathway.Among them,NRF2,an important gene in the NRF2-antioxidant injury signaling,was identified as the target gene of miR-144-3p(2)The upregulation of miR-144-3p was closely related to the decline of antioxidant capacity and the aggravation of oxidative injury in erythrocyteCompared with the control group,the expression level of miR-144-3p was significantly up-regulated in the erythrocytes of HAPC patients and rat models(all,p<0.05).Further correlation analysis showed that miR-144-3p in erythrocytes was significantly positively correlated with ROS and MDA(all,p<0.01),and negatively correlated with antioxidant indexes SOD and GSH(all,p<0.01)(3)miR-144-3p might weaken the antioxidant capacity of erythroid cells by inhibiting NRF2The overexpression of miR-144-3p in K562 cell,could significantly down-regulate the expression levels of antioxidant SOD1,CAT,GCLC,GCLM,GPX1 and NQO1 mRNA in cell,and aggravate the oxidative injury of cell(all,p<0.01).The suppressing expression of miR-144-3p in K562 cell,could significantly up-regulate the expression levels of antioxidant SOD1,CAT,GCLC,GCLM,GPX1 and NQO1 mRNA in cell,and alleviate the oxidative injury of cell(all,p<0.01).Double luciferase reporter assay confirmed that NRF2 was the target gene of miR-144-3p(p<0.01).Conclusion:(1)microRNA expression profiles between AMS patients and those participants without AMS are significantly different,and differential microRNAs are mainly involved in regulating signaling pathways,such as hypoxia adaptation,energy metabolism,angiogenesis and inflammatory response;Inadequate expression of miR-181b-5p leads to reduced inhibition of PRKCD,an important gene of the NF-?B signaling pathway,subsequently increasing peripheral inflammatory response and neuroinflammatory injury,which might contribute to AMS pathogenesis(2)The combination of plasma miR-1183,miR-15b-5p and miR-23b-5p at sea level constitutes a novel biomarker for predicting the AMS risk of young Chinese Han males;The combination of salivary miR-134-3p and miR-15b-5p at sea level,can be used as a novel and non-invasive biomarker for predicting the AMS risk of young Chinese Han males(3)microRNA expression profiles in HAPC patients and healthy controls are significantly different,and differential microRNAs involved in regulating multiple signaling pathways;miR-144-3p inhibits NRF2-antioxidant injury signaling pathway,thus,weakening the antioxidant capacity of erythroid cells,leading to the aggravation of oxidative damage and dysfunction of erythroid cells,which might participate in the pathogenesis of HAPC. |
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