Effect Of EPAS1 On Erythroid Differentiation Of K562 Cell Line Under Hypoxia

Objective To investigate the role of endothelial PAS domain-containing protein 1(EPAS1)gene encoding hypoxia-inducible factor 2?(HIF-2?)in erythroid differentiation of K562human erythroleukemia cells under hypoxic conditions.Methods 1.Erythroid induction and differentiation level detection of K562 cells in hypoxia and normoxia:K562 cells were induced to differentiate into erythroid by using hemin and cytarabine under normoxia(20%O₂)and hypoxia(5%O₂)conditions.The ratio of CD235a⁺CD71⁺cells was detected by flow cytometry,the percentage of hemoglobin-positive cells was detected by benzidine staining,the level of cell proliferation was detected by CCK-8assay,and the m RNA and protein levels of EPAS1,IRS2 and erythroid differentiation related genes were detected by real-time quantitative PCR and Wester blot analysis.2.Lentivirus transfection:We obtained K562 cells with knockdown of EPAS1 gene by lentiviral transfection,and the changes in the degree of erythroid differentiation of these cells1 were detected by the methods above.3.Statistical methods:SPSS 19.0 software was used for data processing,and the data conforming to the normal distribution were expressed as mean±standard deviation(?±SD).One-way analysis of variance(ANOVA)was used for comparison between multiple groups,and LSD test was used for comparison between two groups.P<0.05 was considered statistically significant.Results 1.The proportion of CD235a⁺CD71⁺cells in K562 cells increased significantly under hypoxia[hypoxia(43.40±1.25%)vs.normoxia(34.60±1.18%),n=3,P<0.05].The proportion of CD235a⁺CD71⁺cells in hypoxia induced group was significantly higher than that in normoxia induced group[hypoxia induced group(82.73±1.68%)vs.normoxia induced group(49.73±2.10%),n=3,P<0.05].After knockdown of EPAS1,the proportion of CD235a⁺CD71⁺cells in hypoxia-induced cells decreased[sh-EPAS1(31.36±1.45%)vs.sh-cont(76.43±2.91%),n=3,P<0.05].2.The positive rate of benzidine in hypoxia induced group was significantly higher than that in normoxia induced group[hypoxia induced group(46.4±1.60%)vs.normoxia induced group(19.20±1.41%),n=3,P<0.05].After knockdown of EPAS1,the positive rate of benzidine staining in hypoxia induced group decreased significantly,[sh-cont(31.52±1.30%)vs.sh-EPAS1(19.03±2.01%),n=3,P<0.05].3.After knockdown of EPAS1,the proliferation of K562 cells under normoxia and hypoxia decreased significantly(P<0.05).4.Hypoxia significantly promoted the expression of EPAS1,CD235a and?-globin in K562cells(P<0.05)..After knocking down EPAS1,The expression of CD235a and?-globin decreased significantly(P<0.05).5.Hypoxia could significantly promote the expression of IRS2 in K562 cells(P<0.05).Knockdown of EPAS1 decreased the expression of IRS2 in K562 cells(P<0.05).Inhibition of IRS2 decreased the proportion of CD235a⁺CD71⁺in K562 cells and the expression of CD235a and?-globin gene.Conclusion(1)Hypoxia can up-regulate the expression level of EPAS1 in K562 cells and promote cells to differentiate into erythroid cells.Induction of K562 cells under hypoxia can significantly improve the degree of erythroid differentiation.(2)Knockdown of EPAS1 inhibits the induction of erythroid differentiation of K562 cells under normoxia and hypoxia.EPAS1 is an important factor regulating erythroid differentiation of K562 cells.(3)Hypoxia can up-regulate the expression of IRS2 in K562 cells,the level of IRS2 is positively regulated by EPAS1,and EPAS1 can mediate the erythroid differentiation of K562by regulating the level of IRS2.