Hypoxia Promotes the Molecular Mechanism of High-Altitude Polycythemia by Mediating the Demethylation of Hepcidin mRNA through HIF-1α
Objective:To investigate the roles of HIF-1α and Hepcidin in the pathogenesis of high-alti-tude polycythemia(HAP).Methods:Peripheral blood hemoglobin concentration[Hb],hemoglobin mass(Hbmass),blood volume,and peripheral blood O2 saturation(SpO2)were measured in subjects with high-altitude polycythemia(HAE group),healthy individuals residing at the same high altitude(HH group),and healthy individuals born at high altitude but residing at low altitude for over 2 years(LH group).ELISA was used to determine the levels of HIF-1α and Hepcidin in peripheral blood serum.HepG2 cells were cultured under hypoxia in vitro,with experimental groups including Control and Hypoxia groups.The expression levels of HIF-1α and Hepcidin mRNA and protein were measured.m6A content analysis,RNA immunoprecipitation(RIP),dual-luciferase reporter assay,and mRNA stability analysis were used to explore the molecular mech-anisms by which HIF-1α mediates m6A demethylation of Hepcidin mRNA.Results:Compared with the LH group,the HH group had increased levels of[Hb],Hbmass,and blood volume,and decreased SpO2;com-pared with the HH group,the HAE group had increased levels of[Hb],Hbmass,and blood volume,and de-creased SpO2(P＜0.05).Compared with the Control group,the Hypoxia group showed increased relative ex-pression levels of HIF-1α mRNA and protein,and decreased relative expression levels of Hepcidin mRNA and protein in HepG2 cells(P＜0.05).The m6A(%)level in the Hypoxia group was lower than in the Con-trol group(P＜0.05).mRNA stability analysis showed that the relative expression level of Hepcidin mRNA in the Hypoxia group was lower than in the Control group(P＜0.05).Dual-luciferase reporter assay showed that relative luciferase activity was higher in HepG2 cells co-transfected with Hepcidin 3'-UTR and HIF-1α shR-NA compared to cells co-transfected with Hepcidin 3'-UTR and shRNA NT(P＜0.05).RIP results showed that the enrichment level of Hepcidin 3'-UTR was higher in the FTO Ab group compared to the IgG group(P＜0.05).Conclusion:Hypoxia-induced upregulation of HIF-1α mediates the demethylation of Hepcidin mR-NA,contributing to the progression of high-altitude polycythemia.
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