Bone Marrow Transplantation Center, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310003, China.
Institute of Hematology, Zhejiang University, Hangzhou, China.
BMC Cancer. 2021 Jan 7;21(1):36. doi: 10.1186/s12885-020-07744-x.
Isocitrate dehydrogenase (IDH1/2) gene mutations are the most frequently observed mutations in cartilaginous tumors. The mutant IDH causes elevation in the levels of R-enantiomer of 2-hydroxylglutarate (R-2HG). Mesenchymal stromal cells (MSCs) are reasonable precursor cell candidates of cartilaginous tumors. This study aimed to investigate the effect of oncometabolite R-2HG on MSCs.
Human bone marrow MSCs treated with or without R-2HG at concentrations 0.1 to 1.5 mM were used for experiments. Cell Counting Kit-8 was used to detect the proliferation of MSCs. To determine the effects of R-2HG on MSC differentiation, cells were cultured in osteogenic, chondrogenic and adipogenic medium. Specific staining approaches were performed and differentiation-related genes were quantified. Furthermore, DNA methylation status was explored by Illumina array-based arrays. Real-time PCR was applied to examine the signaling component mRNAs involved in.
R-2HG showed no influence on the proliferation of human MSCs. R-2HG blocked osteogenic differentiation, whereas promoted adipogenic differentiation of MSCs in a dose-dependent manner. R-2HG inhibited chondrogenic differentiation of MSCs, but increased the expression of genes related to chondrocyte hypertrophy in a lower concentration (1.0 mM). Moreover, R-2HG induced a pronounced DNA hypermethylation state of MSC. R-2HG also improved promotor methylation of lineage-specific genes during osteogenic and chondrogenic differentiation. In addition, R-2HG induced hypermethylation and decreased the mRNA levels of SHH, GLI1and GLI2, indicating Sonic Hedgehog (Shh) signaling inhibition.
The oncometabolite R-2HG dysregulated the chondrogenic and osteogenic differentiation of MSCs possibly via induction of DNA hypermethylation, improving the role of R-2HG in cartilaginous tumor development.
异柠檬酸脱氢酶（IDH1/2）基因突变是软骨肿瘤中最常见的突变。突变 IDH 导致 R-（-）-2-羟基戊二酸（R-2HG）的水平升高。间充质基质细胞（MSCs）是软骨肿瘤的合理前体细胞候选物。本研究旨在探讨代谢物 R-2HG 对 MSCs 的影响。
用浓度为 0.1 至 1.5 mM 的 R-2HG 处理或不处理人骨髓 MSCs 进行实验。细胞计数试剂盒-8 用于检测 MSCs 的增殖。为了确定 R-2HG 对 MSC 分化的影响，将细胞培养在成骨、软骨和成脂培养基中。进行特定的染色方法，并定量分化相关基因。此外，通过基于 Illumina 阵列的阵列探索 DNA 甲基化状态。实时 PCR 用于检测涉及的信号成分 mRNAs。
R-2HG 对人 MSCs 的增殖没有影响。R-2HG 以剂量依赖性方式阻断成骨分化，而促进 MSC 成脂分化。R-2HG 抑制 MSC 软骨分化，但在较低浓度（1.0 mM）下增加与软骨细胞肥大相关的基因表达。此外，R-2HG 诱导 MSC 明显的 DNA 高甲基化状态。R-2HG 还改善了成骨和成软骨分化过程中谱系特异性基因的启动子甲基化。此外，R-2HG 诱导了 SHH、GLI1 和 GLI2 的启动子甲基化和 mRNA 水平降低，表明 Sonic Hedgehog（Shh）信号通路受到抑制。
代谢物 R-2HG 通过诱导 DNA 高甲基化，改变 MSCs 的软骨和成骨分化，从而可能在软骨肿瘤的发展中发挥作用。
