葡萄糖转运相关基因SLC2A4RG在脑胶质瘤发生过程中的分子机制研究

Glioma is the most frequent and devastating primary brain tumor resulted in environmental and genetic factors. While the majority of malignant tumors (including brain tumor) cells on glucose consumption were significantly increased. As glucose transport carriers, the relationship of the tumor and GLUT had become hot topics recently. SLC2A4RG, the transcript regulator of GLUT4, plays an essential role in helping GLUT4 transfer from vesicle inside the cell to cytomembrane and promote the cell absorption of glucose. In our previous study, we have identified a susceptive SNP (46C/T) of glioma, which is located on the 3'UTR of gene SLC2A4RG. We observed that the person with CC genotype had a significantly increase risk of glioma than whom with TT genotype (OR = 1.92). Further suggest that SCL2A4RG play an important role in the occurrence of glioma. This project aims through experimental study on function of SLC2A4R gene and 3'UTR of 46C/T susceptibility mechanism for polymorphic loci in glioma, thus systematic, comprehensive state SLC2A4RG in the mechanism of cerebral tumorigenesis of glioma, and provide new clues or targets for the treatment of glioma.

脑胶质瘤是遗传与环境因素共同作用的恶性肿瘤。脑胶质瘤等恶性肿瘤对葡萄糖消耗量明显增加，葡萄糖转运载体GLUT家族与肿瘤的关系是近来研究的热点，SLC2A4RG是葡萄糖转运蛋白4 （GLUT4）的转录增强调节子，我们前期的精细定位研究发现SLC2A4RG基因的3'UTR的46C/T是脑胶质瘤易感基因多态位点，CC基因型个体患脑胶质瘤的风险显著升高(OR=1.92)，国际TCGA数据库显示SLC2A4RG在脑胶质瘤患者组织中显著高表达；进一步提示SCL2A4RG是脑胶质瘤的发生过程中起重要作用。本项目拟通过过表达或干扰SCL2A4R的细胞表型实验研究基因的功能，探索参与葡萄糖异常摄取从而导致肿瘤的分子机制和3'UTR的46C/T 多态位点易感机制，从而较深入、全面的阐明SLC2A4RG在脑胶质瘤发生过程中的作用机制，为脑胶质瘤的治疗提供新的线索或靶标。

实验室之前的分子流行病学研究工作发现SLC2A4RG基因3’UTR 46 T>C多态位点和脑胶质瘤发生发展显著相关（P = 1.26E-11），我们通过双荧光素酶系统检测到相对于T等位型，C等位型能显著降低含有SLC2A4RG基因3’UTR的荧光素酶报告基因的表达。在临床组织样本中的定量RT-PCR实验显示：相对于17例对照样本，在52例脑胶质瘤样本中SLC2A4RG基因的mRNA水平没有显著变化。而在包括16例对照样本和273例脑胶质瘤样本的组织芯片中进行的SLC2A4RG蛋白的免疫组化实验显示：胞核SLC2A4RG蛋白在脑胶质瘤组织中有着显著性的下调，并且随着脑胶质瘤级别的升高而逐渐下降。随后我们提取12例正常对照样本和24例脑胶质瘤样本中的胞核蛋白，通过Western blot验证这一结果；发现相对于正常对照样本，脑胶质瘤样本中胞核SLC2A4RG蛋白有着显著性的下调，同时也随着脑胶质瘤级别的升高而逐渐下降。通过CCK-8和克隆形成率实验，检测到过表达SLC2A4RG可以抑制细胞的增殖能力；而干扰SLC2A4RG可以促进细胞的增殖能力。流式细胞仪检测过表达SLC2A4RG后，细胞周期被阻滞在G2/M期；Western blot 检测发现过表达SLC2A4RG后，G2/M细胞周期相关蛋白Cyclin B和CDK1表达水平显著下调。通过 ChIP 和荧光素酶报告基因实验发现SLC2A4RG可以和Caspase-3、Caspase-6 启动子上潜在结合位点直接结合影响SLC2A4RG对Caspase-3和Caspase-6的转录调控，并证实BAG6和14-3-3降低了SLC2A4RG对Caspase-3和Caspase-6的激活调控。首次提出了SLC2A4RG在脑胶质瘤中的发生发展及预后中的重要作用，将为后续的脑胶质瘤机制研究和临床治疗提供参考。