The Research On DNA Methylation Profiles And The Effect Of Related Genes MICU1 In High Altitude Adaptation

Part ?:DNA METHYLATION PROFILES OF TIBETAN AND CHINESE IN THE QINGHAI-TIBET PLATEAUBackground:Environmental hypoxia,low temperatures,low precipitation and high ultraviolet radiation are the harsh nature of the Tibetan Plateau.Compared with other native and immigrant high-altitude residents,Tibetans have higher ventilation shape parameter values,hypoxia ventilatory response,and higher arterial oxygen saturation,lower hemoglobin concentration,and better sleep quality at night.It is reported that the Tibetans possess levels of endothelial nitric oxide synthase than Plain people,which could explain the improved oxygen diffusion and blood flow in their lungs.In addition,Tibetans possess heritable adaptations to hypoxic environments,as indicated by birth weight,and oxygen saturation of blood and hemoglobin levels in infants and adults after exercise.All these factors allow Tibetans to live at high altitudes In Tibetans,the adaptation to high altitudes might have genetic components.Simonson et al found that the haplotypes of EGLN1 and PPARA were significantly positively associated with decreased hemoglobin B phenotype that is unique to Tibetans.Beall et al.and Yi et al.reported that EPAS1 was a candidate gene that had undergone positive selection for high-altitude adaptation.Simonson et al.,Wang et al.and Yang et al.demonstrated that EDNRA and YES1 were involved in high-altitude adaptation.However,the association between gene expression at DNA methylation level and hypoxia has seldom been reported.Thienpontet al.reported that tumor hypoxia reduces the activity of oxygen-dependent ten eleven translocation enzymes,which catalyze DNA demethylation through 5-methylcytosine oxidation.This suggests that methylation is associated with hypoxia.Dasgupta et al.provided evidence of hypoxia causing heightened promoter methylation.SUBJECTS AND METHODS:In this study,we recruited 10 high-altitude Tibetans(5 males and 5 females;average age:27.5 years),10 low-altitude Tibetans(5 males and 5 females;average age:29.9 years)Ten people moved to the high-altitude Han(5 males and 5 females;average age:36.8 years)and 10 ordinary Hans(5 males and 5 females;average age:30.2 years).Using a microarray-based approach to identify differential methylation regions(DMRs)between the Tibetan and immigrant Hans,and analyzing differentially methylated genes through the Kyoto Encyclopedia of Genes and Genomics(KEGG)Path Enrichment Analysis(IPA)and gene function enrichment analysis(Go)for its function.RESULTS:KEGG analysis of differential methylation sites suggested that high-altitude Tibetans and low-altitude Tibetans were enriched in arrhythmogenic right ventricular cardiomyopathy,hypertrophic cardiomyopathy,and dilated cardiomyopathy signaling pathways.The high-altitude Han and low-altitude Han residents are uniquely enriched in the insulin signaling pathway and the mTOR signaling pathway.The KEGG analysis at the promoter level suggests that the glycolysis/glycogen heterogeneous pathway is unique to high-altitude Tibetans and low-altitude Tibetans.CONCLUSIONS:In the current study,we used an array-based approach to uncover genome-wide DNA methylation profiles inresident Plateau Tibetans versus resident Plain Tibetans and migrant Plateau Hans versus resident Plain Hans.Moreover,typical functional annotation analysis was performed on the corresponding differentially methylated genes in the differentially methylated sites and the promoter level.The present study provides valuable information for exploring the epigenetic regulation of DNA methylation in resident Plateau Tibetans versus resident Plain Tibetans and migrant Plateau Hans versus resident Plain Hans,which may provide a new field in understanding the mechanism of high-altitude adaptation.Part ?:DNA METHYLATION PROFILES OF TIBETAN AND CHINESE IN THE QINGHAI-TIBET PLATEAUOBJECTIVE:To study changes in the cerebrovascular reactivity(CVR)at different altitude area in healthy adults.SUBJECTS AND METHODS:CVR was tested using transcranial Doppler combined with C02 inhalation,near-infrared spectroscopy(NIRS)was used to detect the regional cerebral oxygen saturation(rSc02).Blood samples were collected,and the vasoactive substances in serum were detected using the enzyme-linked immunosorbent assay.In this study,59 healthy adults were divided into 3 groups:low altitude group,medium altitude group and high altitude group.All the indicators in low altitude group were tested at 24h before departure and after arrival from Beijing(at an altitude of 44.4 m)to Xining(at a medium altitude of 2200 m).Then,after resting for 48h,all the indicators were tested at 24h and 48h after arrival from Xining(at a medium altitude of 2200 m)to Yushu Jiegu town(at a high altitude of 3700 m)together with those at the medium altitude.Intergroup comparisons were made for the subjects in the three altitudes.RESULTS:There was an increase in the CVR in low altitude group after acute exposure to high altitude,and the difference was significant(CVR:1.94re was vs.0.91 ±0.53,p<0.001);the CVR index was increased,and the difference was significant[cerebrovascular reserve index(CVRI):3.65he CVR vs.1.37e CVR,p<0.001];the rSc02 level was decreased with the increase of altitude,and the difference was significant[(66.78±4.61)%vs.(70.29±4.52)%,p<0.001].The levels of vasoactive substances in low altitude group were decreased after acute exposure to high altitude compared with those before exposure:NO:[(79.14±9.54)?mol/L vs.(58.01±9.93)?mol/L,p<0.001];serum eNOS level was increased,and the difference was significant[(77.23±6.20)pg/ml vs.(65.07±9.82)pg/ml,p<0.001];EPO:[(84.68±13.16)pg/ml vs.(65.01±5.92)pg/ml,p<0.001];VEGF:[(71.91±11.62)pg/ml vs.(54.92±11.86)pg/ml,p<0.001];sFlt:[(384.18±42.73)pg/ml vs.(320.62±78.96)pg/ml,p<0.001].There was also an increase in CVR in medium altitude group after acute exposure to high altitude,and the difference was significant[CVR:2.00±0.79 vs.0.91 ±0.66,p<0.001];the difference of CVRI was significant[3.83±0.67 vs.1.67±0.87,p<0.001];rSc02 was slightly decreased with the increase of altitude,and the difference was not statistically significant[(67.53±4.61)%vs.(69.63±5.59)%,p<0.001].Before exposure to high altitude area,the levels of NO,NOS,EPO,VEGF,and sFlt in low and medium altitude groups were higher than those in high altitude group.CVR level of subjects at different altitudes were negatively related to the Sc02(r=-0.91)but positively related to NO and NOS levels(rs=0.89,r=0.75);CVR was moderately related to VEGF and EPO(rs=0.45,r=0.42).rSc02 was positively related to RBC,HB and VEGF levels(r=0.89,r=0.75,rs=0.86),but had a moderately negative correlation with NO and NOS levels(rs=-0.52,r=-0.57).CONCLUSIONS:After subjects at a low altitude are exposed to high altitude rapidly,CVR is increased,RBC and vasoactive substances in serum,such as NO,eNOS,and EPO,are dramatically increased,VEGF is increased first and then decreased,sFlt-1 level is increased gradually,and rSc02 level is gradually decreased with the increase of altitude,indicating the local brain anoxia of subjects at a high altitude.Part ?:FUNCTIONAL STUDY OF THE PLATEAU ADAPTIVE GENE MICU1 DURING HMATOPOIETIC DIFFERENTIATIONBackground:According to the results of previous studies,the DNA methylation profile of Tibetan and Han genomic DNA was used to analyze the pathways enriched by differential methylation genes.We selected calcium signaling pathway and hypoxia adaptation related mitochondrial calcium ion uptake protein 1 for related experiments.the study.Hypoxia induces hypoxia-inducible factor HIF activation to decrease mitochondrial membrane potential,and activated HIF further activates a range of factors that cause mitochondrial autophagy.Mitochondrial membrane potential is reduced by autophagosomes,which induces mitochondrial autophagy.Mitochondrial dysfunction leads to imbalance of calcium homeostasis,which will cause autophagy and death.Mitochondrial calcium ion uptake protein 1(MICU1),a mitochondrial calcium ion unidirectional transport complex(MCU,MICU1,MICU1),is an important molecule that maintains mitochondrial calcium homeostasis and is a negative regulator of mitochondria-dependent death pathways.The study found that the expression level of MCUR1 was significantly increased after humans entered the plateau,suggesting that MCUR1 may play a role in adapting to the plateau environment.So what is the function of MICU1 in the complex?To this end,we constructed a K562 cell line stably knocking down MICU1,and identified the expression of the vector,and explored the function of MIUC1 on erythroid differentiation under hypoxia and its possible mechanism.To explore the differentiation of MIUC1 into erythroid in hypoxic environment.Regulation in the process and its effect on proliferation,differentiation and apoptosis of K562.Methods:The expression of MICU1 protein in K562 cell line was detected by Western blot.K562 cells were packaged with lentiviral technology to stably knock down the expression of MICU1,cultured in normoxia and hypoxia.The knockdown effect of MICU1 and the expression of erythroid differentiation markers cd235a and ?-globin were detected by qRT-PCR.Westernblot was used after reaching the standard.The expression of MIUC1,P53,BAX and Bcl-2 was detected.The proliferation of K562 cells after knockdown of MICU1 was detected by CCK8 method.The cycle and apoptosis of K562 cell line after knockdown of MICU1 were detected by ANNEXINV-APC/7-ADD.Results:Hypoxia inhibited the expression of CD35a and ?-globin in K562 cells and inhibited erythroid differentiation.The results of CCK-8 indicated that knockdown of MICU1 significantly inhibited the proliferation of K562 cells.The results of flow cytometry showed that knockdown of MCUR1 could be performed.Promote apoptosis of K562 cells;Western blotting experiments showed that knockdown of MICU1 promoted the expression of apoptosis-related proteins p53 and BAX and inhibited the expression of BCL-2.These results suggest that MICU1 can promote the proliferation of K562,inhibit the apoptosis of K562,and promote the differentiation of erythroid.Conclusion:Our results indicate that in the K562 cell model,MICU1 can regulate K562 differentiation into erythroid cells,regulate proliferation,differentiation and apoptosis,and its regulation of K562 differentiation and apoptosis may be achieved by regulating the expression of MICU1.This further confirmed a possible new mechanism by which MIUC1 hypoxia induces erythroid differentiation.