Nrf2调控食管癌干细胞自我更新的作用及其机制研究

Esophageal cancer is one of the most lethal cancers worldwide. Recently, somatic mutations of Nrf2 were found in esophageal cancer. It was also found that Nrf2 is related to the regulation of self-renewal in embryonic and adult stem cells. However, the role of Nrf2 in esophageal cancer stem-like cells remains unclear. In our previous work, we found that Nrf2 was highly expressed in the enriched esophageal cancer stem-like cells by gene chip. Therefore, we hypothesize that Nrf2 is a key regulator in esophageal cancer stem-like cells. In this study, we will examine the Nrf2 expression levels in the esophageal cancer stem-like cells enriched by ALDH sorting or SP sorting, verify the regulatory functions of Nrf2 in self-renewal, proliferation and chemoresistance by in vitro and in vivo experiments, and clarify the underlying molecular mechanisms. We will examine the downstream targets of Nrf2 and the effect of ROS level on self-renewal in esophageal cancer stem-like cells. These findings will reveal the functions and mechanisms of Nrf2 in the self-renewal of esophageal cancer stem-like cells.

食管癌恶性程度高且患者预后差。近年研究发现Nrf2在食管癌中存在基因突变，同时Nrf2也被发现参与胚胎干细胞和成体干细胞的自我更新调控。然而，Nrf2通路与食管癌干细胞的关系尚未被阐明。我们在前期工作中利用基因芯片发现Nrf2在富集的食管癌干细胞中呈异常高表达，并由此提出科学假说：Nrf2是食管癌干细胞自我更新的关键调控因子。本课题将围绕Nrf2调控食管癌干细胞自我更新的作用和机制开展深入研究：采用流式分选富集食管癌干细胞，使用实时荧光定量PCR和Western Blot检测Nrf2及下游通路分子的表达水平，通过体内体外实验验证Nrf2对食管癌干细胞自我更新、增殖和药物敏感性的调控作用；并进一步阐明Nrf2通路调控食管癌干细胞自我更新的机制，明确其下游关键基因，同时揭示Nrf2调控的活性氧水平改变对食管癌干细胞自我更新的影响，从而最终阐明Nrf2在食管癌干细胞中的重要作用及其分子机制。

食管癌恶性程度高、预后差，是我国高发恶性肿瘤。近年研究发现Nrf2在食管癌中存在基因突变，同时发现Nrf2参与干细胞的调控。然而，Nrf2通路与食管癌干细胞的关系尚未被阐明。本研究围绕Nrf2在食管癌干细胞自我更新的作用和机制开展深入研究。采用流式分选法富集的食管癌干细胞中Nrf2及下游通路分子异常高表达而ROS水平较低。在食管癌细胞系中采用特异性抑制剂及慢病毒等方法，明确Nrf2可调控自我更新和增殖的作用和机制，抑制Nrf2可增加食管癌细胞对化疗药物顺铂的敏感性。构建食管类器官模型，明确Nrf2与其调控因子CUL3在具有自我更新和干性的基底层细胞中存在特殊高表达模式。分析Nrf2和下游关键基因及氧化应激标志物在食管癌临床标本中的异常表达模式，Nrf2在肿瘤浸润前缘呈现强阳且在基底样食管癌中表达明显升高，且存在表达定位不同。采用高深度肿瘤相关基因高通量测序发现Nrf2异常表达的机制与其基因关键结合抑制功能域突变和发生基因拷贝数扩增有关。进一步用三种不同方式获得的食管癌干细胞和非干细胞进行DNA甲基化谱研究，分析Nrf2和氧化应激通路、信号传导通路及代谢通路等基因的甲基化差异。综上，本研究揭示了Nrf2在食管癌及肿瘤干细胞中的重要作用及其分子机制，为其应用于食管癌的防治奠定理论基础。