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OACSTPCD
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O2 饱和度(SpO2).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Hepci-din 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,SpO2 降低;与 HH 组相比,HAE 组[Hb]、Hb-mass水平和血容量升高,SpO2 降低(P＜0.05).与 Control 组相比,Hypoxia 组 HIF-1α mRNA 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-1α shRNA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 mR-NA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-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.
白洁;黄河;张莲;浩光东;万玛草
联勤保障部队第940 医院,甘肃 兰州 730030
高原红细胞增多症缺氧诱导因子-1α铁调素m6A RNA去甲基化修饰
High-altitude polycythemiaHypoxia-inducible factor-1αHepcidinm6A RNA demethylation
《河北医学》 2024 (008)
1250-1255 / 6
甘肃省自然科学基金,(编号:22JR11RA008);第九四〇医院院内课题,(编号:2022yxky005)
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