CPEB4调控Bcl-2影响脑胶质瘤放疗敏感性的分子机制及临床意义研究

Radioresistance is the crucial problem in the therapy of glioma, indentify and make validation of the protein maker with sensitivity of radiation and explain the details of the mechanism will bring a new and valuable choice for glioma therapy.CPEB4 has been proved to play a key role in the translational control of cancer progression. Our previously work indicated that high expression of CPEB4 is significantly corrrelated with the poor prognosis of glioma patients who underwent radiotherapy after operation. Functional experiments showed that, inhibited the expression of CPEB4 will enhance the sensitivity of radioation of U251-MG; meanwhile, the Bcl-2 also been reduced after the CPEB4 was knockdown in U251-MG;in additional, expression of CPEB4 in U251-MG was increased following the explosion to radiatin, and the CPEB4 was also translocated with the mitochondria location lable protein Cytochrome C. Therefore, we make hypothesis: CPEB4 is required for Bcl-2 induced apoptosis to regulate the sensitivity of radiation of glioma. In this project, we will collect more details to prove the relationship between CPEB4 and Bcl-2, and the mechanism of CPEB4 regulate apoptosis to effect the sensitivity of radiation of glioma, we will also make validation of CPEB4 and the downstream protein between the prognosis of glioma patients who received radiotherapy after operation. This will provide new knowledge to solve the problem on radioresistance and provide new diagnosis parameter for sensitivity prediction, and disclose the mechanism of the CPEB4 regulate the radio-sensitivity of glioma may provide a new alternative for glioma therapy.

CPEB4在多种肿瘤的发生发展中起关键作用。而CPEB4是否影响脑胶质瘤放疗敏感性尚不明确。我们预实验发现CPEB4在放疗抵抗病人的胶质瘤标本中表达明显增加，敲低CPEB4的表达可明显抑制胶质瘤细胞的放疗抗性;敲低CPEB4的表达， 凋亡关键蛋白Bcl-2的表达也出现下调；此外，放疗后CPEB4在胶质瘤细胞中发生明显上调，并且转位至线粒体。因此，我们推测：胶质瘤细胞放疗后通过上调CPEB4的表达与Bcl-2等抗凋亡分子发挥互作而抑制线粒体介导的细胞凋亡，进而帮助肿瘤细胞逃避射线损伤。为了阐明其中的具体机制，我们将进行后续实验获取CPEB4与Bcl-2发生互作的证据，确证CPEB4调控凋亡机制影响胶质瘤放疗敏感性的具体环节，进一步在临床样本中验证CPEB4及其调控靶标蛋白与术后放疗的胶质瘤患者的预后关系，最终为临床开发判断放疗敏感性的诊断指标和攻克胶质瘤提供理论依据。

CPEB4作为多种实体肿瘤的关键调控分子参与了脑胶质瘤放疗抵抗的表型形成过程。本课通过对前期采集的临床手术标本进行免疫组化染色与临床关键指标的生物统计学分析，发现CPEB4高表达组病人其预后不良；采用多变量分析方法，比较了年龄、别性、术前KPS评分、2007 WHO分级、肿瘤大小、肿瘤部位、术前是否存在颅内压增高及是否存在癫痫症状等临床指标，发现CPEB4的表达水平与脑胶质瘤病人的年龄、性别及肿瘤部位无关，然而，CPEB4的高表达却与脑胶质瘤的术前KPS评分不良、2007WHO分级增高、肿瘤直径大、术前颅内压增高及存在癫痫症状密切相关（P <0.05）；ROC曲线分析方法提示，与传统判断肿瘤预后的指标KPS、肿瘤大小、是否全切、WHO 分级比较， CPEB4表达水平的变化可以作为判断星形脑胶质的预后指标之一；COX回归模型多变量分析提示，CPEB4表达水平可以作为判断星形胶质瘤预后的一个独立指标；进一步的亚组分析发现，接受术后放疗的人脑星形细胞瘤患者，CPEB4高表达组的术后总生存时间明显短于低表达组。为了进一步探讨CPEB4影响脑胶质瘤放疗抵抗的分子机制，我们首先采用si-hRNA的方法敲减了CPEB4在星形胶质瘤细胞U251-MG中的表达，利用生长曲线、平板克隆的方法检测了敲减CPEB4以后胶质瘤细胞的生物行为改变，结果提示，敲减CPEB4在胶质瘤细胞中的表达虽然不能引起体外肿瘤细胞增殖能力的改变，但是敲减CPEB4的表达可以明显抑制胶质瘤细胞的侵袭、克隆形成能力；我们对不同剂量X射线剂量处理的胶质瘤细胞的蛋白样本进行检测，发现，在较低射线剂量范围内CPEB4在胶质瘤细胞中的表达随照射剂量的增加而增加，这提示CPEB4在胶质瘤细胞接受射线处理后的细胞应激过程中可能发挥关键作用；为了验证CPEB4是否可以在胶质瘤细胞接受射线处理后发挥促进射线抗拒的作用，我们对CPEB4敲减前后的胶质瘤细胞进行了X射线处理，发现敲减CPEB4的表达可以明显增强胶质瘤细胞对放疗的敏感性；流式细胞学检测发现，敲减CPEB4的表达可以明显增加放疗引起的胶质瘤细胞的凋亡。