The Function And Molecular Mechanism Of The Candidate High-Altitude Adaptation-Related HOXA9 Gene

Objective: To explore how HOXA9 and its polymorphism in the Tibetan population participate in the high-altitude adaptation.Methods: The expression levels of HOXA9 under hypoxic conditions were assessed using real-time quantitative polymerase chain reaction(RT-qPCR)and Western blotting(WB)assays.The vectors stably knock down or overexpress wild-type or mutant HOXA9 were constructed in K562 cell line.Hemin was used to induce K562 cells to differentiate into erythroid cells under hypoxic conditions,and then the expression changes of erythroid markers CD235 a and ?-globin were assessed by RT-qPCR.The benzidine staining assay was used to evaluate the ratio of erythrocyte differentiation.Through chromatin immunoprecipitation(ChIP)-real-time quantitative polymerase chain reaction(RT-qPCR),electrophoretic mobility shift assay(EMSA)and luciferase assays,we analyzed how HOXA9 and its polymorphism regulate the hypoxia-inducible factor HIF1 A,and whether their effects on erythropoiesis are HIF1A-dependent under hypoxic conditions.Results: We observed that the hypoxia induces expression of HOXA9.Knockdown of HOXA9 decreased the levels of the erythroid markers CD235 a and?-globin,and the proportion of erythrocyte differentiation in K562 cells.Conversely,overexpression of wide-type HOXA9(HOXA9-WT)inceased the levels of CD235 a and ?-globin,and the proportion of erythrocyte differentiation in K562 cells.Notably,compared with the HOXA9-WT,overexpression of mutant HOXA9(rs140596580,p.Pro189Thr)reduced the proportion of erythrocyte differentiation,the expression levels of erythroid markers CD235 a and ?-globin.Mechanistically,by browsing the transcription factor target gene database,we observed that HIF1 A is the potential downstream target gene of HOXA9.Then,we observed that knockdown of HOXA9 reduces the HIF1 A mRNA and protein expression levels;while overexpression of HOXA9-WT induces the HIF1 A mRNA and protein expression levels.Next,the specific binding of HOXA9-WT to the HIF1 A first intron region was demonstrated by the EMSA and ChIP-qPCR experiments,and was further confirmed by the luciferase assay that HOXA9-WT can activate the transcription activity of HIF1 A.Whereas,the mutant HOXA9(rs140596580,p.Pro189Thr)lost its promoting effects on transcription activity of HIF1 A because it could not bind to HIF1 A.Finally,we found that the promoting effects on erythropoiesis by HOXA9-WT are abolished when HIF1 A was knocked down,indicating that HOXA9 depends on HIF1 A to regulate erythropoiesis in hypoxia.Conclusion:(1)We demonstrated that the expression levels of HOXA9 are induced by hypoxia in the process of erythroid differentiation;(2)HOXA9induces the erythropoiesis under hypoxic conditions;(3)HOXA9-mediated erythropoiesis under hypoxia is dependent on its transcriptional regulation of HIF1A;(4)HOXA9 polymorphism(rs140596580,p.Pro189Thr)loses its regulation of HIF1 A and thus reduces the erythropoiesis.