去乙酰化酶SIRT3调控线粒体ATP酶介导髓系白血病细胞能量代谢及耐药的机制研究

Recently, a rebirth of the interest in the energetic metabolism of cancer spurred, and mitochondrion has become hotspot issue. Our previous studies have demonstrated, for the first time, the mitochondrial ATPsyn-β downregulation, which represents a novel protein involved in multidrug resistance, indeed plays an important role in drug resistance in myeloid leukemia cells. The epigenetic regulation-DNA methylation plays important role in the dyregulation of mitochondrial ATPsyn-β. However, the use of demethylation methods cannot fully reverse chemotherapy resistance,which means that there are still other epigenetic mechamisms take part in the regulation. Recent studies identified acetylated lysine residues in several subunits of mitochondria ATP synthase and implied that SIRT3 might regulate the mitochondria ATPase activity through deacetylation. Based on previous findings, the purpose of this project is to analyze the regulatory effect of SIRT3 on mitochondrial ATPsyn-β. We will use the techniques such as gene transfection, RNA interference technique, ChIP, Co-IP, and other techniques to explore the possible mechanisms of drug resistance associated with mitochondrial ATPsyn-β expression, especially focus on energetic metabolism and autophagy. This project would help further in searching new potential target from the new energetic metabolism perspective for the reversal of myeloid leukemia multidrug resistance.

线粒体是近年来研究肿瘤细胞能量代谢的热点之一。项目组前期研究首次证实了线粒体ATPsyn-β作为一种新的耐药相关蛋白，与髓系白血病细胞耐药存在密切联系，表观遗传调控DNA甲基化异常在其中发挥了重要作用。然而仅仅使用去甲基化手段并不能完全恢复耐药白血病细胞对化疗药物的敏感性，说明还有其他表观遗传调控机制发挥了作用。新近研究提示组蛋白去乙酰化酶SIRT3可以调节线粒体ATP酶活性。本课题拟在前期基础上，通过基因转染、RNA干扰、ChIP、Co-IP、电镜、荧光显微镜等技术，研究SIRT3对线粒体ATPsyn-β的调控机制，探讨二者介导能量代谢及促进自噬致髓系白血病耐药的作用，以期从能量代谢的新角度为逆转髓系白血病耐药提供新的线索。

本研究在前期研究的基础上旨在探讨SIRT3去乙酰化线粒体ATPsyn-β、通过调控自噬参与慢性髓系白血病细胞（CML）的能量代谢和耐药。通过Co-IP、慢病毒转染、RNAi等方法证实了CML耐药细胞中SIRT3对线粒体ATPsyn-β的乙酰化修饰作用，两者表达呈正相关；通过对耐药白血病细胞SIRT3表达水平/ATPsyn-β对应变化状态及细胞内能量参数的分析发现靶向SIRT3可以影响细胞的能量代谢；SIRT3通过AKT/P13K/mTOR、NF-κB途径介导的CML细胞自噬活性增强促进了白血病细胞的化疗耐药。通过以上实验明确了SIRT3参与慢粒白血病细胞能量代谢、靶向SIRT3促进了白血病细胞对伊马替尼的敏感性，为逆转慢性髓系白血病耐药提供了新的靶标。