Research Center for High Altitude Medicine, Qinghai University, Xining 810001, China; Key Laboratory of Application and Foundation for High Altitude Medicine Research in Qinghai Province, Xining 810001, China.
Research Center for High Altitude Medicine, Qinghai University, Xining 810001, China; Key Laboratory of Application and Foundation for High Altitude Medicine Research in Qinghai Province, Xining 810001, China; Affiliated Hospital of Qinghai University, Xining 810001, China.
Life Sci. 2021 Feb 1;266:118873. doi: 10.1016/j.lfs.2020.118873. Epub 2020 Dec 9.
Hypoxia-inducible factors (HIFs) play important roles in the pathogenesis of erythrocytosis in chronic mountain sickness (CMS). von Hippel-Lindau (VHL) is a key regulator of hypoxia that can direct the poly-ubiquitylation and degradation of HIFs. Epigenetic mechanisms are believed to contribute toward adaption to chronic hypoxia. Here, we investigated the contribution and mechanism of VHL methylation in rats with erythrocytosis in CMS.
The methylation status of VHL was measured via bisulfite sequencing PCR, while VHL, DNMT1, DNMT3α, and DNMT3β expression were assessed using real-time reverse transcription PCR and western blotting. HIF-2α and EPO expression levels in bone marrow were determined via immunohistochemical staining, and erythroid hyperplasia in bone marrow sections were observed with hematoxylin and eosin staining.
We found that chronic hypoxia triggered erythroid hyperplasia in the bone marrow and increased the quantity of peripheral red blood cells in CMS rats. Chronic hypoxia significantly induced methylation at the CpG site in the VHL promoter, decreased VHL expression, and increased HIF-2α and EPO expression. Chronic hypoxia increased DNMT3α and DNMT3β expression, consistent with the decrease in VHL expression. The DNA methyltransferase inhibitor 5-azacytidine reduced chronic hypoxia-induced erythroid proliferation in the bone marrow of rats with CMS by suppressing VHL methylation and DNMTs expression.
Our study suggests that VHL methylation contributes toward excessive erythrocytosis in CMS by upregulating the HIF-2α/EPO pathway in the bone marrow of rats. We demonstrated that the DNMT inhibitor 5-azacytidine can attenuate erythroid hyperplasia in the bone marrow by demethylating the VHL promoter.
缺氧诱导因子（HIFs）在慢性高原病（CMS）引起的红细胞增多症发病机制中起重要作用。von Hippel-Lindau（VHL）是缺氧的关键调节因子，可指导 HIFs 的多泛素化和降解。表观遗传机制被认为有助于适应慢性缺氧。在这里，我们研究了 VHL 甲基化在 CMS 红细胞增多症大鼠中的作用和机制。
通过亚硫酸氢盐测序 PCR 测量 VHL 的甲基化状态，同时通过实时逆转录 PCR 和 Western blot 评估 VHL、DNMT1、DNMT3α 和 DNMT3β 的表达。通过免疫组织化学染色测定骨髓中 HIF-2α 和 EPO 的表达水平，并用苏木精和伊红染色观察骨髓切片中的红细胞增生。
我们发现慢性缺氧会引发 CMS 大鼠骨髓中的红细胞增生，并增加外周血红细胞数量。慢性缺氧显著诱导 VHL 启动子 CpG 位点的甲基化，降低 VHL 表达，并增加 HIF-2α 和 EPO 的表达。慢性缺氧增加了 DNMT3α 和 DNMT3β 的表达，与 VHL 表达的降低一致。DNA 甲基转移酶抑制剂 5-氮杂胞苷通过抑制 VHL 甲基化和 DNMTs 表达，减少 CMS 大鼠慢性缺氧诱导的骨髓红细胞增殖。
我们的研究表明，VHL 甲基化通过上调 CMS 大鼠骨髓中的 HIF-2α/EPO 通路，导致红细胞过度增多。我们证明，DNMT 抑制剂 5-氮杂胞苷可以通过去甲基化 VHL 启动子来减轻骨髓中的红细胞增生。
