The Molecular Mechanism Of EPAS1 Regulating The Proliferation Of Bone Marrow Erythroblasts In Patients With Chronic Mountain Sickness

Chronic Mountain Sickness(CMS)is a clinical syndrome caused by the gradual loss of acclimatization to the high altitude hypoxic environment among people living in high altitude areas.Its main characteristics are excessive erythrocytosis(EE)and hypoxemia,but the pathogenesis of CMS has not been clarified.Early studies on EE mainly focused on up-regulated expression of circulating EPO under hypoxic condition,stimulating hematopoietic activity.However,more and more scholars found that the EPO level in peripheral blood of some CMS patients was similar to that of healthy people at the same altitude.At present,there is no exact explanation for the phenomenon that people with normal EPO levels still continue to produce excess erythropoiesis.Hypoxia inducible factor(HIF)is the center of hypoxic response.The stability of HIFs-α was enhanced during hypoxia,involved in hypoxia response by regulating the expression of downstream target genes.HIF-1α and endothelial PAS domain containing protein-1(EPAS1,which is also known as HIF-2α)have overlapping target genes,but also playing an independent role according to different cell types and oxygen concentrations.For adults,bone marrow is the main site of erythropoiesis.Hypoxia is the initial factor of CMS,but the over reaction of bone marrow to hypoxia is the fundamental cause for EE.Our previous study showed that there was no obvious change in the proportion of CD34+ cells in CMS patients,while the proportion of CD71+ cells increased significantly.Thus,the expression changes of HIF-1α and EPAS1 and their mechanism in bone marrow erythroblasts of patients with CMS are still unclear.Part Ⅰ The Expression of HIF-1α and EPAS1 in Bone Marrow Erythroblasts of Patients with CMSObjective: To evaluate the expression of HIF-1α and EPAS1 in bone marrow erythroblasts of patients with CMS,and to explore the role and significance of hypoxia inducible factor in the excessive accumulation of erythrocytes in CMS.Methods: Bone marrow liquids of patients with CMS and old fractures at the same altitude were collected.Further,density gradient centrifugation,immunomagnetic separation,RT-q PCR and western blotting were used to study the expression of HIF-1 α and EPAS1 in bone marrow erythroblasts of patients with CMS.Results: A total of 21 bone marrow samples of patients with CMS and 14 bone marrow samples of control group were collected.The characteristics of bone marrow cells showed that the ratio of myeloid to erythroid was significantly decreased,and the percentages of polychromatic erythroblasts and orthochromatic erythroblasts were significantly increased in patients with CMS(P < 0.05).There was no difference in HIF-1α expression between the two groups(P > 0.05).The m RNA and protein levels of EPAS1 in CD71+ erythroblasts of patients with CMS increased significantly(P < 0.05).The results of correlation analysis showed that the m RNA level of HIF-2α was positively correlated with RBC count(r = 0.703,P < 0.001),negatively correlated with blood oxygen saturation(r =-0.793,P < 0.001),and positively correlated with hemoglobin(r = 0.826,P < 0.001).Conclusion: EPAS1 may affect the proliferation of erythroblasts in bone marrow,leading to excessive accumulation of erythrocytes in CMS.Part Ⅱ The Effect of EPAS1 on the Proliferation,Cycle and Expression of Erythroid Differentiation Antigen of K562 cells under Chronic HypoxiaObjective: To evaluate the changes of cell proliferation,cell cycle and erythroid differentiation antigen expression after K562 cells were transfected with EPAS1 overexpression / interference lentivirus.Methods:The effects of EPAS1 on K562 cell proliferation and cell cycle were detected by MTS and flow cytometry after transfection of K562 cells by Lv-EPAS1/sh-EPAS1 lentivirus under hypoxic conditions.The expression of K562 erythroid differentiation antigens were also detected by flow cytometry after Hemin induced differentiation of K562 cells under hypoxic conditions.Results: 1.The m RNA and protein levels of EPAS1 were higher in Lv-EPAS1 group(P < 0.05);The m RNA and protein levels of EPAS1 were lower in sh-EPAS1 group(sh-EPAS1-1,sh-EPAS1-2,sh-EPAS1-3),and the expression of sh-EPAS1-1 was the lowest(P < 0.05).2.After overexpression of EPAS1 gene,the proliferation of K562 cells and the number of Brd U positive cells in S phase increased(P < 0.05);the proportion of cells in S phase increased,the proportion of cells in G1 phase decreased(P < 0.05);the proportions of cells in CD235a+and CD71+ CD235a+ decreased(P < 0.05).3.After EPAS1 gene interference,the proliferation of K562 cells and the number of Brd U positive cells in S phase decreased(P < 0.05),the proportion of S phase cells decreased,the proportion of G1 phase cells increased(P < 0.05),the proportions of CD235a+ and CD71+ CD235a+ cells increased(P < 0.05).Conclusions:Under chronic hypoxia,EPAS1 promote the proliferation of CD71+ erythroblasts.EPAS1 promotes the proliferation of erythroblasts at CD71 stage under chronic hypoxia.Part Ⅲ The Study of EPAS1 on Regulating the Downstream Gene Pathway of Erythroid Proliferation and Differentiation under Chronic HypoxiaObjective: The molecular mechanism of EPAS1 in the development and differentiation of erythroblasts under chronic hypoxia was studied by transcriptome sequencing using Illumina sequencing platform.Methods: The overexpression group(Lv-EPAS1,HE),interference group(sh-EPAS1,HEi)and control group(Lv-NC,HC1;sh-NC,HC2),induced by hemin for 72 hours under hypoxia,were collected to construct NEB library.We proceeded to sequence on Illumina sequencing platform to study the mechanism of EPAS1 in the differentiation and development of erythroblasts.Results: 1.By veen map analysis of differential genes,86 differential genes with the same trend,regulated by EPAS1,were screened,participating in the regulation mechanism of erythroid hematopoiesis.2.Go function enrichment found that the top 15 differential genes were mainly involved in cell proliferation,differentiation and hemoglobin synthesis.3.KEGG pathway enrichment found that some differential genes of the top 20 pathway were annotated to erythroid proliferation and differentiation related pathways,such as PI3K-Akt、Rap1 pathways.4.Combined with the enrichment of Go and KEGG,we screened four erythroid candidate genes: FLT1,SOCS1,STAT3,EBP42.Conclusions: EPAS1 participates in the process of proliferation,differentiation and development of erythroblasts by regulating the erythroid related gene “FLT1/SOCS1/STAT3/EPB42”.