地西他滨通过下调TIGAR介导髓系白血病细胞凋亡的机制研究

Decitabine (DAC) has been found to exert anti-metabolic and anticancer activities in myeloid leukemia cells in vitro and in vivo. Generation of reactive oxygen species (ROS) has been reported to induce myeloid leukemia cell apoptosis. The exact mechanism of DAC induced ROS accumulation in leukemia cells is unclear. TIGAR is a p53-target gene, which inhibits glycolysis and apoptosis. In our previous study, high level of glycolytic metabolism and an increased TIGAR gene transcription level have been found in patients with myeloid leukemia at diagnosis. TIGAR can protects K562 cells from apoptosis, and TIGAR gene silencing induces apoptosis in myeloid leukemia cells through ROS accumulation. We found DAC downregulated TIGAR expression and induced apoptosis in leukemia cells. We hypothesized that DAC regulates metabolic pathways and promotes ROS accumulation leading to myeliod cell apoptosis through downregulation of TIGAR expression. To investigate this hypothesis, the methods such as real time PCR, Western blot, viral vector transfection, RNA interference were used to demonstrate the regulation mechanism of DAC downregulating TIGAR expresion on myeloid cells. To clarify the exact mechanism of ROS accumulation, cell metabolism, and the relationship between them.

去甲基化药物地西他滨（DAC）能诱导髓细胞白血病细胞凋亡，文献报道DAC诱导细胞凋亡与活性氧（ROS）生成相关，但确切机制尚不清楚。TIGAR是p53调节线粒体呼吸的靶基因，具有抑制糖酵解和抗凋亡作用。我们前期研究发现髓细胞白血病患者发病时为糖酵解代谢，TIGAR呈高表达状态；TIGAR高表达能保护K562细胞免于凋亡，沉默TIGAR促进ROS生成致白血病细胞凋亡。DAC能下调TIGAR表达，诱导白血病细胞凋亡。为此我们提出假说，DAC通过下调 TIGAR 基因表达，调控细胞内能量代谢途径，促进ROS生成，诱导髓细胞白血病细胞凋亡。为验证这假说，我们通过K562、HL-60细胞和原代细胞，采用real time PCR、Western blot、病毒载体转染、RNA干扰等手段，旨在明确DAC下调TIGAR及其作用机制，明确DAC调控ROS生成和细胞能量代谢的作用，以及两作用间的相关性。

髓系白血病包括急性髓细胞白血病（AML）和慢性髓细胞白血病（CML），是血液系统最常见的恶性疾病之一，其发病机制复杂。研究者发现去甲基化药物地西他滨（DAC）对白血病细胞的真正作用机制远比之前所认为的复杂。DAC能诱导髓细胞白血病细胞凋亡，文献报道DAC诱导细胞凋亡，与活性氧（ROS）生成相关，但确切机制尚不清楚。髓细胞白血病细胞存在着异常能量代谢，TIGAR是p53调节线粒体呼吸的靶基因，具有抑制糖酵解和抗凋亡作用。.本课题组前期研究发现（PLoS One.2010;5: e13186；J Hematol Oncol. 2016;9(1):128.）髓细胞白血病患者发病时为糖酵解代谢，TIGAR呈高表达状态；TIGAR高表达能保护K562细胞免于凋亡，沉默TIGAR促进ROS生成致白血病细胞凋亡。DAC能下调TIGAR表达，诱导白血病细胞凋亡。我们通过HL-60，K562细胞系、原代细胞，分别从real time PCR、Western blot、腺病毒载体转染、RNA干扰等手段，从分子和细胞等多方面探讨DAC在髓细胞白血病中的机制作用。.TIGAR 具有抑制细胞糖酵解和抗凋亡的作用。DAC 可以通过 ROS 增高诱导髓细胞白血病细胞凋亡。我们的前期结果证实了体内外髓细胞白血病细胞均存在 TIGAR 高表达；无氧糖酵解能诱导 TIGAR 高表达，降低细胞内ROS，从而达到抗凋亡作用。而我们发现 DAC 可以下调 TIGAR 表达，并诱导髓细胞白血病细胞凋亡。DAC诱导ROS水平上升，与糖酵解途径相关，细胞凋亡增加。能量代谢参与DAC促进细胞凋亡。DAC诱导ROS增高受TIGAR基因调控。DAC通过细胞内糖酵解流量增加，PPP 途径流量下降，细胞内 NADPH 减少，细胞内 ROS 上调，促进细胞凋亡。DAC诱导ROS的作用依赖DNMT1。DAC掺入到细胞DNA过程中对DNA有损害作用，DAC下调TIGAR表达促进细胞凋亡是通过线粒体途径。