IDH1基因突变与PI3K/AKT/mTOR信号通路激活在脑胶质瘤肿瘤发生中的作用机理研究

Recently, isocitrate dehydrogenase-1 (IDH1) somatic mutations were detected in the majority of (more than 80%) human brain gliomas including astrocytoma(WHO Grade II),oligodendroglioma(Grade II), anaplastic astrocytoma (Grade III) and secondary glioblastoma(Grade IV). IDH1 mutation is an early event in the development of brain gliomas, and is an important prognostic biomarker in gliomas. In a preliminary study, we found the PI3K/AKT/mTOR pathway is frequently activated in gliomas harboring IDH1 mutation. However, it is not very clear whether the activation of PI3K/AKT/mTOR pathway is the cause or consequence of IDH1 mutations in gliomas. And the relationship between IDH1 mutation and the activation of PI3K/AKT/mTOR pathway involved in the tumorigenesis of gliomas need to be elucidated in our research. In this study, we will detect the IDH1 and IDH2 somatic mutations ,and PI3K/AKT/mTOR pathway in a large number of glioma samples in China at first. The second, we plan to establish an IDH1 mutant (for example, R132H, R132C, R132L or R132S) knockin mice model by using the protocol of TP53 point mutation knockin mice, and they will be carefully examined histologically and genetically after brain tumors develop in these IDH1 mutant knockin mice. Genetic analysis will be carried out by next generation sequencing, to identify additional genetic alterations which cooperate with IDH1 mutations. Cell lines will be established from brain tumors developed in IDH1 mutant knockin mice. The third, the expression of genes in PI3K/AKT/mTOR pathway will be analyzed in the IDH1 knockin mice, the relationship between IDH1 mutation and the activation of PI3K/AKT/mTOR pathway will be clarified in vivo. In pararell, some cell lines, such as A735 human astrocyte cell line, CRL-2032 mouse cell line and human CHG-5 (WHO Grade II), CB193 (Grade III), U251 and U87MG glioma (Grade IV), will be over-expressed IDH1 point mutant sequences by using gene transfection, then the cell cycle, apoptosis and growth patterns of the treated cells will be analyzed using flow cytometry, MTT analysis and scanning electron microscopy, also Western Blotting will be performed to detect the activation of PI3K/AKT/mTOR pathway in the treated cell lines. Metabolic products in IDH1 mutant tumor cells will be monitored by gas chromatography/mass spectrometry. Finally, the role of IDH1 mutation and activation of PI3K/AKT/mTOR pathway involved in the tumorigenesis of brain gliomas will be elucidated in partly in vitro and in vivo study.

近年来研究显示：异柠檬酸脱氢酶-1 (IDH1)基因在II和III级脑胶质瘤和继发性胶质母细胞瘤(IV级)中有很高的突变频率(>80%)，IDH1基因突变是脑胶质瘤肿瘤发生过程中的早期事件。我们的前期研究发现：在IDH1突变的胶质瘤患者，有极高的PI3K/AKT信号通路激活现象，而IDH1突变后能否通过影响PI3K/AKT通路参与肿瘤发生尚不清楚。因此本研究拟建立人-鼠嵌合型IDH1点突变基因敲入小鼠模型，观察小鼠脑瘤的发生及其表型；同时在正常胶质细胞系与胶质瘤细胞系中过表达突变型IDH1基因，通过流式细胞仪等进行细胞周期与凋亡分析，气相色谱检测代谢产物变化，并对在胶质瘤发生与进展中起重要作用的PI3K/AKT信号通路激活与调控作用进行研究，明确IDH1基因突变对正常与肿瘤细胞生物学性状的影响，解析该信号通路与IDH1突变的密切联系，以期阐明IDH1基因突变在脑胶质瘤肿瘤发生中的作用机理。

近年来研究发现，脑胶质瘤患者存在高频率的异柠檬酸脱氢酶-1（Isocitrate dehydrogenase-1, IDH1）基因突变现象，且该突变与脑胶质瘤的诊断分型和临床预后有明确的关系，对其深入研究，有望找到脑胶质瘤治疗的新靶点，对改善当前脑胶质瘤治疗现状有着深远的意义。本研究着重于阐明IDH1基因突变在脑胶质瘤发生发展中的作用机制，具体包括：IDH1突变与PI3K/Akt/mTOR信号通路激活之间的关系，IDH1突变和 MGMT基因启动子甲基化之间的关联度，IDH1下游靶分子HIF-1α在脑胶质瘤对替莫唑胺耐药性中的作用，微小RNA（miRNA）与IDH1突变胶质瘤进展及预后之间的关系，及通过构建转基因动物模型探索IDH1基因突变与胶质瘤发生之间的关系。重要结果包括：IDH1 R132H的突变可促进磷酸化Akt显著上调，提示IDH1突变可激活PI3K信号通路；相关性分析发现IDH1 突变和MGMT 基因启动子甲基化存在显著关联；HIF-1a敲低后阻断由TMZ处理引起的MGMT表达上调及Notch1信号通路激活，进而增强胶质瘤细胞对TMZ的敏感性；通过采用miRNAs芯片对不同级别脑胶质瘤（WHOII、III、IV级各3例）进行芯片筛选发现13个差异表达的microRNA分子，其中7个属于致癌miR-17-92 (miR-17, miR-19a, miR-20a, and miR-92-1)和miR-106b-25 (miR-106b, miR-93 and miR-25) 簇，这表明不同级别脑胶质瘤之间差异性表达的miRNAs在脑胶质瘤发生及进展中发挥着重要作用，可作为分子标记物帮助对胶质瘤分类及预后进行判断；生物信息学（miRNA-seq及miRNA芯片）筛选及搜集临床样本进行测序并qRT-PCR验证发现，miR-155在IDH1突变的低级别胶质瘤及胶质母细胞瘤中表达均显著下调；生存分析发现miR-155过表达是影响患者预后的危险因素；甲基化芯片结果分析及细胞学研究发现IDH1基因突变可通过基因组甲基化引起miR155宿主基因甲基化，引起miR155表达下调；IDH1基因突变可引起胶质瘤细胞增殖受到抑制，过表达miR155则可促进胶质瘤细胞的增殖。以上研究揭示IDH1基因突变脑胶质瘤发生发展，以及患者预后中发挥着极其重要的作用。