胶质瘤IDH突变对乙酰辅酶A相关代谢的影响

为系统研究IDH 突变对胶质瘤细胞主要代谢过程和代谢物的影响，项目将使用液相色谱-质谱技术，分析胶质瘤组织内IDH1/2 的R132 位点突变情况；再利用各种色谱-质谱联用技术对以乙酰辅酶A 为核心的糖酵解、三羧酸循环、磷酸戊糖途径、尿素循环、核苷、氨基酸和脂肪酸等胞内主要代谢过程代谢物进行定量分析，阐明乙酰辅酶A相关主要代谢途径中代谢物的量与R132 位点突变和肿瘤病理分级之间的关系；从中筛选出受突变影响最显著的代谢物；构造IDH1(R132H)突变细胞，在不同氧供条件下进行数字基因表达谱分析和定量代谢组学分析，验证组织代谢组学分析的发现，阐明最显著变化代谢物对乙酰辅酶A 代谢及与缺氧诱导因子（HIF）表达程度、稳定性和降解的关系；干预筛选到的显著变化代谢物的胞内浓度，研究其对肿瘤细胞侵袭性、生长、抗凋亡特性等恶性生物学行为的影响，揭示IDH突变引起的代谢物变化在胶质瘤发生发展中的作用。

In order to study the effects on cell metabolism from mutant IDH in glioma， IDH1/2 R132 mutation analysis will be performed by liquid chromatography - mass spectrometry (LC-MS) for glioma tissues. Then, LC-MS, gas chromatography - mass spectrometry and capillary electrophoresis - mass spectrometry will be employed to quantify metabolites involved in glycolysis, TCA cycle, PPP, urea cycle, nucleotides, amino acids and fatty acids metabolism in view of metabolomics. Metabolites severely affected by IDH mutation will be defined. Subsequently, U251 constructs with R132H mutation will be cultured under normoxia and hypoxia conditions. Their transcriptome will be analyzed by using DGE. Simultaneously, metabolomics analysis will be performed to quantify the acetyl CoA related metabolites. The aim is to elucidate the relationship of significantly changed metabolites, acetyl CoA, 4 oncometabolites and hypoxia induciable factors (HIFs), especially the stability and degradation of HIFs. Effects on U251 R132H growth, apoptosis, invasion and relevant biological traits will be evaluated through perturbating the identified significantly changed metabolites by IDH mutation. The purpose is to functionally elucidate the mechanism of IDH affected metabolites in oncogenesis and to provide a holistic view of effects from mutant IDH on the glioma metabolism.