线粒体自噬NIX和Pink1信号交互作用对索拉非尼治疗肝癌的影响及干预研究

The effect of general autophagy on hepatoma cells survival/death outcome remains controversial and unclear, which restricts the therapeutic efficacy of sorafenib in HCC. Further probe into selective mitophagy and tumor treatment facilitation emerges as new hot spot. Based on preliminary studies and emerging new clues, we propose new ideas: NIX and Pink1, mitochondrial proteins, may interact and dominate different mitophagy modes in hepatoma cell lines. We assume that IRE1/NIX activation promotes sorafenib-resistance, on the contrary, induction of Pink1-dependent cell death signals sensitizes cell response to sorafenib, which results in an enhanced lethality. Herein, we will first observe the occurrence and development characteristics of mitophagy in cells dynamically. And then, in vitro study will be performed to explore the influence of NIX and Pink1 interactive expression on mitophagic hepatoma cells outcome. Further research will be organized to investigate the mechanisms of how endoplasmic reticulum stress affect the BNIP3/NIX and Pink1/Parkin activities. Finally, we will validate and elucidate this impact on sorafenib-based HCC therapy with targeted regulation of NIX and/or Pink1-dependent mitophagy in vivo. If this proposal is granted and work smoothly, it will offer elicitations to extricating us from the clinical predicament and bring insights into a novel and potential HCC therapeutic strategy in the future.

肝癌细胞发生广义自噬后生死转归机制依然不清，制约着肝癌关键用药索拉非尼的临床疗效，通过探讨选择性线粒体自噬促进治疗成为研究新热点。基于前期工作及浮现新线索，我们提出新设想：线粒体蛋白NIX和Pink1可能交互作用且共同主宰肝癌细胞线粒体自噬不同作用模式；IRE1/NIX激活可能代表索拉非尼耐药机制；通过诱导Pink1细胞致死信号，可促进肝癌细胞应答索拉非尼的敏感性。为此，我们首先在细胞分子水平动态观察线粒体自噬的发生、发展特征，然后通过体外干预，研究NIX和Pink1交互作用对发生线粒体自噬的肝癌细胞转归影响，同时探讨影响BNIP3/NIX和Pink1/Parkin信号通路活动的内质网应激调节机制；最后从整体水平靶向调节NIX和/或Pink1依赖性线粒体自噬水平，考证其对以索拉非尼为基础的肝癌药物治疗方案的影响。预期所获研究数据可为摆脱索拉非尼临床治疗困境提供新思路和新靶点。

肝癌细胞自噬后细胞生死转归不详，已发现制约着肝癌一线用药索拉非尼的临床疗效，其中NIX及PINK1介导线粒体自噬参与肝癌细胞命运选择成为研究热点。我们在国家自然科学基金课题的资助下，首先观察发现索拉非尼可诱导肝癌细胞线粒体自噬增多及显著影响线粒体蛋白NIX、PINK1的表达，提示NIX及PINK1介导线粒体自噬对肝癌细胞生死转归发挥不同作用；其次，通过体外干预研究发现上调NIX蛋白表达可以诱导肝癌细胞自噬，继而降低肝癌细胞对索拉非尼的敏感性；进一步通过构建耐索拉非尼药物肝癌细胞发现线粒体NIX呈高表达且诱导细胞自噬，通过基因干预敲减NIX后可降低细胞自噬并增加耐药细胞对索拉非尼的敏感性，阐明线粒体蛋白NIX介导细胞自噬参与索拉非尼耐药机制；第三，我们从离体到整体水平进一步考证了线粒体蛋白NIX介导细胞自噬对肝癌治疗的影响，通过基因干预、药物干预发现NIX高表达肝癌模型瘤体增大明显且索拉非尼抑瘤率降低，提示了NIX具有“促癌”作用且参与肝癌耐药过程。第四，通过基因干预上调PINK1蛋白，我们发现可诱导细胞自噬，并增加肝癌细胞死亡率、凋亡率，抑制肝癌细胞的增殖和克隆形成能力，提示索拉非尼通过线粒体PINK1调控肝癌细胞自噬性细胞死亡。实验数据不仅丰富了索拉非尼耐药机制，且对肝癌治疗中自噬抑制剂使用提供了可靠的参考依据。发表论文10篇，参加国内外学术交流2次，培养研究生9人。