低氧通过HIF-1α介导Hepcidin mRNA的去甲基化修饰促进高原红细胞增多症的分子机制研究

Hypoxia Promotes the Molecular Mechanism of High-Altitude Polycythemia by Mediating the Demethylation of Hepcidin mRNA through HIF-1α

目的:探讨HIF-1α和Hepcidin在高原红细胞增多症致病机制中的作用。方法:招募和测定高原红细胞增多症(HAE组)和同一高海拔地区健康个体(HH组),以及出生于同一高海拔地区并在超过2年的时间段居住于低海拔地区的健康个体(LH组)外周血血红蛋白浓度[Hb]、血红蛋白质量(Hbmass)、血容量和外周血O_(2)饱和度(SpO_(2))。ELISA法测定上述个体外周血清HIF-1α和Hepcidin的水平。体外低氧诱导HepG2细胞,实验分组为Control组和Hypoxia组,测定细胞中HIF-1α和Hepcidin mRNA和蛋白质表达水平。m6A含量分析、RNA免疫共沉淀法(RIP)、双荧光素酶报告基因分析法,mRNA稳定性分析法深入探讨HIF-1α对Hepcidin mRNA的m6A去甲基化修饰的分子机制。结果:与LH组相比,HH组[Hb]、Hbmass水平和血容量升高,SpO_(2)降低;与HH组相比,HAE组[Hb]、Hbmass水平和血容量升高,SpO_(2)降低(P<0.05)。与Control组相比,Hypoxia组HIF-1αmRNA和蛋白质相对表达水平升高,Hepcidin mRNA和蛋白质相对表达水平降低(P<0.05)。与Control组相比,Hypoxia组HepG2细胞的m6A(%)水平降低(P<0.05)。mRNA稳定性分析HepG2细胞中Hepcidin mRNA水平,与Control组相比,Hypoxia组Hepcidin mRNA相对表达水平降低(P<0.05)。双荧光素酶报告基因分析结果显示,与共转染了Hepcidin 3'-UTR和shRNA NT组的HepG2细胞相比,共转染了Hepcidin 3'-UTR和HIF-1αshRNA组的HepG2细胞相对荧光素酶活性升高(P<0.05)。RIP结果显示,与IgG组相比,FTO Ab组Hepcidin 3'-UTR富集水平升高(P<0.05)。结论:低氧上调HIF-1α介导Hepcidin mRNA的去甲基化修饰参与高原红细胞增多症疾病进展。

Objective:To investigate the roles of HIF-1αand Hepcidin in the pathogenesis of high-altitude polycythemia(HAP).Methods:Peripheral blood hemoglobin concentration[Hb],hemoglobin mass(Hbmass),blood volume,and peripheral blood O_(2) saturation(SpO_(2))were measured in subjects with highaltitude 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-1αand 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-1αand 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 mechanisms by which HIF-1αmediates 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 SpO_(2);compared with the HH group,the HAE group had increased levels of[Hb],Hbmass,and blood volume,and de-creased SpO_(2)(P<0.05).Compared with the Control group,the Hypoxia group showed increased relative ex-pression levels of HIF-1αmRNA 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-1αshR-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-1αmediates the demethylation of Hepcidin mR-NA,contributing to the progression of high-altitude polycythemia.