自噬相关因子Atg7调控RIP1介导细胞程序性坏死的机制研究

Necrosis and autophagic cell death were the main mode of non-apoptotic cell death and played an important role in cell death. Current studies have confirmed that necrosis could be induced by TNF-α+z-VAD: phosphorylation of serine/threonine kinase 1 (RIP1) binds to RIP3 to form a necrotic complex to initiate necrosis; it was also found that autophagy was enhanced in this process. However, the role of autophagy and its relationship with programmed necrosis is still unclear. Our previous work found that autophagy inhibitor chloroquine or silencing autophagy-related factor Atg7 could inhibit TNF-α+z-VAD-induced cell death, and Atg7 and RIP1 directly bind, suggesting that Atg7-mediated autophagic death was origin of necrosis; Atg7 may be involved in the activation of necrotic signaling pathway by regulating the formation of a necrotic complex by RIP1. In this project, a hypothesis was that necrosis was a type II autophagic cell death, with Atg7 regulating RIP1-RIP3 necrosis complex as an entry point, and autophagy-necrosis complex-programmed necrosis as the overall research idea, analysis of association between autophagic cell death and necrosis provides a new theoretical basis for cancer treatment.

程序性坏死和自噬性死亡是细胞非凋亡死亡的主要方式，在细胞死亡过程中发挥重要作用。目前研究证实程序性坏死可以被TNF-α+z-VAD诱导：丝氨酸/苏氨酸激酶1（RIP1）磷酸化后与RIP3结合形成坏死复合体启动坏死进程；同时发现该进程中自噬增强，但自噬的作用及其与程序性坏死的关系尚不清楚。我们前期工作发现自噬抑制剂氯喹或沉默自噬相关因子Atg7可抑制TNF-α+z-VAD诱导的细胞死亡，并且Atg7与RIP1可直接结合，提示Atg7介导的自噬性死亡是程序性坏死的起源；Atg7可能通过调控RIP1影响坏死复合体的形成，参与程序性坏死信号通路的激活。本项目以程序性坏死是Ⅱ型自噬性死亡为假说，以Atg7调控RIP1-RIP3坏死复合体形成为切入点，以自噬-坏死复合体-程序性坏死为整体研究思路，深入解析自噬性死亡-程序性坏死之间的关联性，为肿瘤治疗提供新的理论依据。

程序性坏死和自噬性死亡是细胞非凋亡死亡的主要方式，在细胞死亡过程中均发挥重要作用。以往的研究发现细胞程序性坏死过程中，经常伴随有自噬的发生，但两者的相关性尚不清楚。我们工作的主要内容包括明确TNF-α+z-VAD诱导细胞程序性坏死过程中，自噬的改变；明确在刺激条件下，细胞内自噬和程序性坏死的因果关系；探索自噬行为改变后，对细胞程序性坏死的影响；探索自噬关键蛋白Atg7和P62对细胞程序性坏死影响的机制。在本项目中，我们证明TNF-α+z-VAD诱导细胞程序性坏死发生的过程中，促进自噬发生；抑制自噬通路后，坏死复合物RIPK1/RIPK3积蓄，p-RIPK1/p-RIPK3表达增加，细胞死亡增加；自噬通路关键蛋白P62通过结合RIPK1和RIPK3，促进其降解从而阻止程序性坏死的发生。总之，这些结果提示在TNF-α+z-VAD刺激条件下，自噬抑制细胞程序性坏死的发生，为肿瘤治疗提供新的理论依据。