IDH1 R132H突变体通过下调p53促进胶质瘤发生的机理

In recent years IDH1 R132H mutation has been found in up to 75% of low grade gliomas. However the mechanism how this mutation leads to tumorigenesis in glioma remains to be clarified. The progress to address this mechanism has been seriously retarded for the reason that no IDH1 R132H knock-in mouse was generated before. To dissect this mechanism, we created conditional knock-in mouse of IDH1 R132H mutant and did some preliminary work. Intriguingly, we found that IDH1 R132H mutant could robustly down regulate the protein level of tumor suppressor gene p53 and further promote poliferation of primary MEF cells isolated from knock-in mouse by virtues of western blot, morphology and cell growth curve.Consistently, glioma samples from patients with IDH1 R132H mutation also showed dramatically down-regulated p53 protein level,indicating that this mutation promotes tumorigenesis in glioma by negatively regulating p53. In this project, we will try to clarify the mechanism how p53 protein level is down regulated in cells expressing IDH1 R132H mutant, and then to investigate whether and how IDH1 R132H mutant influences cell cycle progression, senescence and proliferation of IDH1 R132H knock-in cells. Moreover,we will create glioma mice model by knocking-in IDH1 R132H mutant in brain tissue and collect glioma samples from patients to further dissect the mechanism by which IDH1 R132H leads to tumorigenesis in glioma via down regulating p53 protein level. The performance of this project may provide new clue for the treatment of glioma.

近几年在高达75%的Ⅱ、Ⅲ级胶质瘤中发现异柠檬酸脱氢酶1（IDH1）发生了R132H突变。而该突变体导致胶质瘤发生的机理还不甚清楚。因为没有动物模型，目前的研究主要限于体外培养的细胞，严重制约了研究进展。为此，我们构建了IDH1 R132H突变体的条件性基因敲入小鼠，并用此小鼠对该突变体导致肿瘤发生的机理进行了初步研究。通过Western blot、形态学及绘制细胞生长曲线，我们发现该突变体能强烈下调抑癌因子p53的蛋白水平并促进细胞增殖；临床胶质瘤标本的检测结果也表明该突变体能下调p53。说明下调p53可能是其引起胶质瘤发生的主要原因之一。本项目拟首先研究该突变体下调p53的分子机制；进而研究其对细胞周期、细胞衰老及细胞增殖的影响；最后通过在小鼠脑组织表达该突变体建立小鼠胶质瘤模型，并结合对临床胶质瘤标本的研究揭示该突变体通过下调p53导致胶质瘤发生的机理，为胶质瘤的治疗提供新的思路。

异柠檬酸脱氢酶1（IDH1）R132H突变在多种肿瘤中被发现而受到广泛的关注，其致癌机理还有待阐明。p53是目前人类发现的最重要的抑癌基因之一，p53的失活与多种肿瘤的发生密切相关。然而IDH1 R132H突变体是否对p53有直接的调节作用目前仍不清楚。本研究的前期工作中发现该突变体能强烈下调抑癌因子p53的蛋白水平并促进细胞增殖，说明下调p53可能是其引起胶质瘤发生的主要原因之一。本项目首先研究了该突变体下调p53的分子机制；进而研究了该突变体下调p53在肿瘤发生中的作用；最后结合临床胶质瘤标本对机理进行了验证。需要注意的是我们尽了最大努力去构建突变体条件性敲入的胶质瘤模型，但我们没有得到模型小鼠，因为突变体会引起脑出血导致胚胎致死。.总之，在本项目的研究中，我们先构建了肝脏特异性IDH1 R132H突变体的基因敲入小鼠，并分离了胚胎成纤维细胞。通过Western blot、Real-time PCR和荧光素酶报告实验，我们发现IDH1 R132H突变体通过产生2-HG抑制了HIF-2α的羟基化及其泛素化降解导致HIF-2α升高，累积的HIF-2α促进了miRNA-380-5p的转录。miR-380-5p直接作用于p53 mRNA的3’-UTR，使其降解。最后，进一步用实验证明IDH1 R132H突变体下调p53显著促进了细胞增殖和抵抗由DNA损伤诱导的细胞凋亡，促进了胶质瘤的发生。本项目的研究揭示了IDH1R132H突变体下调p53促进胶质瘤发生的分子机理，加深了目前对胶质瘤发生的理解，为胶质瘤的治疗提供新的思路。