缺氧微环境下RLIP76介导的HIF-1α甲基化修饰对胶质瘤细胞氧化应激敏感性的实验研究

Hypoxia is a hall mark of glioma cells and this stress significantly change the sensitivity of glioma cells to oxidative stress, which is important to tumorgenesis. Previous studies showed that RLIP76 plays a critical role in oxidative stress of cancer, however, the relationship of the hypoxia-induced oxidative stress and RLIP76 in glioma cells remains unknown. We have demonstrated that RLIP76 functions as an oncogene through PI3K pathway in glioma cells. In addition, PI3K pathway can affect the methylation modification of the histone lysine methyltransferase (KMT). Our previous studies revealed that downregulation of RLIP76 leads to preventing HIF-1α methylation under hypoxia conditions and HIF-1α stability plays a critical role in regulating oxidative stress of tumor. This newly identified RLIP76-mediated methylation regulation of HIF-1α stability may contribute the sensitivity of hypoxia-induced oxidative stress in glioma cells and ultimately promote tumor to adaptation to the hypoxia microenvironment. In this study we will explore the effect of RLIP76 on oxidative stress in glioma and its regulatory mechanism under hypoxia environment. Furthermore, we will investigate whether RLIP76 mediates the sensitivity of glioma cells to hypoxia-induced oxidative stress by methylation-dependent regulation of HIF-1α stability. It is first time to connect RLIP76, tumor microenvironment and oxidative stress in glioma cells and provide a brand new idea for understanding the regulatory mechanism of tumor oxidative stress in hypoxia microenvironment.

缺氧是胶质瘤体内微环境的重要特征，缺氧诱导下肿瘤的氧化应激能力发生改变，这对肿瘤发生发展至关重要。RLIP76是调节细胞氧化应激的中间枢纽，然而缺氧下RLIP76在胶质瘤氧化应激中的意义目前尚不清楚。申请人前期研究显示RLIP76可通过调控PI3K通路促进胶质瘤恶变，而PI3K通路可影响组蛋白赖氨酸甲基转移酶KMT的甲基化修饰能力。项目预实验提示缺氧环境下改变RLIP76表达可影响HIF-1α甲基化从而改变其稳定性，HIF-1α稳定性是调控肿瘤氧化应激的关键信号。因此我们推测RLIP76可能经PI3K通路调控KMT活性从而影响HIF-1α甲基化修饰，最终使得胶质瘤细胞氧化应激耐受性增加从而适应体内缺氧微环境导致恶变。本项目将明确缺氧环境下RLIP76对胶质瘤氧化应激的作用并揭示RLIP76调控HIF-1α甲基化修饰影响氧化应激的具体分子机制，为理解缺氧环境下肿瘤氧化应激调控提供新思路。

人脑胶质瘤作为典型乏氧肿瘤，缺氧诱导的氧化应激是胶质瘤的主要特征，也是促进肿瘤发生发展的重要机制。本项目研究发现：在缺氧胶质瘤细胞模型中，RLIP76表达可引起肿瘤细胞内ROS水平增高、线粒体膜电位降低从而导致胶质瘤细胞氧化应激水平升高并降低肿瘤对氧化应激的耐受性，由此在低氧环境下促进胶质瘤细胞的细胞增殖、迁移并降低肿瘤细胞的凋亡。利用PI3K激动剂激活PI3K-AKT信号可逆转在缺氧微环境下因抑制RLIP76表达对胶质瘤的抗肿瘤作用，说明PI3K-AKT信号通路在RLIP76的抗氧化应激损伤中发挥重要作用。RLIP76激活PI3K-AKT经典通路的同时，可以抑制HIF-1α的甲基化修饰，从而激活HIF-1α下游信号，提示RLIP76对胶质瘤细胞的氧化应激保护其机制可能与激活PI3K-AKT信号及HIF-1α甲基化有关。此外，PI3K-AKT经典通路激活后促进组蛋白赖氨酸甲基转移酶6（KMT6）磷酸化，上调HIF-1α的表达。而低氧环境下在敲除RLIP76的胶质瘤中过表达KMT6后，能逆转RLIP76介导的HIF-1α甲基化作用,说明RLIP76介导的HIF-1α甲基化修饰是通过PI3K-AKT调控的KMT6发挥作用。在小鼠胶质瘤模型中，发现抑制RLIP76表达后荷瘤小鼠肿瘤体积明显缩小，并减少肿瘤HIF-1α、GPx、LDHA、GLUT1的表达，提示RLIP76介导的HIF-1α信号通路在促进胶质瘤氧化应激敏感性及能量代谢中发挥作用。本研究深入探讨了RLIP76介导的HIF-1α甲基化途径对于胶质瘤氧化应激的调控作用及其分子机制，为胶质瘤的治疗提供新的思路和靶点。项目研究成果在Oncology Reports期刊发表论文1篇，中华医学会全国神经外科年会中发表会议论文2篇。