RITA诱导的p53信号通路激活及其在人脑胶质瘤细胞凋亡中的作用机制研究

RITA is a potential tumor suppressor molecule and its mechanism is controversial, especially has not been reported in the study of brain glioma therapy. We obtained the deduction that the correlation of RITA-MDM4 is the highest in three interaction of RITA with p53/MDM2/MDM4 the previous work is based. In this study, RITA ,as a MDM4 potential inhibitor, is simulated optimization with MDM4 by molecular dynamics（ MD） first ,then tested by In vitro and in vivo experiments. In vitro experiments, in four brain glioma cell lines, we analysis the state of p53 detected mRNA level, evaluate the effect of induction of antitumor activity of brain glioma cell cycle arrest and apoptosis in the activation of p53 pathway with these parameters: p53/MDM4 protein levels, cell viability, cell cycle, apoptosis and soft agar colony formation test index of RITA in biology. In vitro, the successful construction of mouse model with transplanted tumor is the first before drug treatment, then that we detect pathology detection, in situ apoptosis detection and quantitative analysis. The study provide new theoretical basis for RITA pharmacological effects, high activity of tumor suppressor molecule mechanism; and render new practical references for drug therapy of brain glioma; accelerate its clinical application and promote the innovative cancer drugs.

RITA作为潜在的抑癌小分子，其作用机制尚存在争议,我们前期研究发现p53/MDM2/MDM4三个靶蛋白中RITA与MDM4互作相关性最高，其对肿瘤细胞的调控作用及分子机制尚有待进一步证实。本项目首先采用分子动力学计算研究MDM4与RITA的互作关系，NMR证实后，以人脑胶质瘤细胞为研究对象,分析RITA对p53状态、mRNA水平的调控作用，评估相关蛋白水平、细胞活力、细胞周期、细胞凋亡和体外克隆形成能力等指标，进一步研究RITA对脑胶质瘤细胞荷瘤小鼠的治疗，通过病理学检测、原位细胞凋亡检测与定量分析等技术观察其作用效果，旨在阐明RITA-MDM4作用对激活p53信号通路所发挥的关键调控作用及其在恢复野生型p53功能、激活p53通路诱导细胞凋亡的分子机制,探索其能否作为潜在的抗肿瘤小分子药物用于临床治疗，以期为RITA高活性抑癌分子机理阐明提供新的理论依据,为脑胶质瘤的药物治疗提供新参考。

RITA作为抑癌小分子化合物作用机制存在争议，尚未见在脑胶质瘤中试验研究。为了进一步证实RITA与MDMX蛋白之间的互作关系，阐述RITA的作用机理，本项目从分子模拟-细胞-动物水平进行整合性研究。首先采用分子模拟和分子动力学计算研究MDM4与RITA的互作关系；然后以人脑胶质瘤细胞为研究对象,分析RITA对p53状态、mRNA水平的调控作用，评估相关蛋白水平、细胞活力、细胞周期、细胞凋亡和体外克隆形成能力等指标；并在体内研究了RITA对脑胶质瘤细胞荷瘤小鼠的治疗，通过病理学检测、原位细胞凋亡检测与定量分析等技术观察其作用效果。研究结果表明，RITA-MDM4作用对激活p53信号通路发挥了关键调控作用，RITA在恢复野生型p53功能、激活p53通路诱导细胞凋亡上效果显著。小分子RITA在脑胶质瘤中能恢复野生型p53功能、激活p53通路诱导细胞凋亡的分子机制为其用于前期临床治疗，为脑胶质瘤的药物治疗提供了新参考。完成研究论文3篇，其中英文文章1篇，另外1篇SCI论文审稿中。