转录调控因子Nupr1在非小细胞肺癌细胞中的自噬调控机制

The relationship between autophagy and tumorigenesis as well as metastasis is a topic of great interest in clinical research field of tumor molecular biology. Transcriptional regulator Nupr1 has been shown to be related to the regulation of autophagy pathway with less clear mechanism. We found that Nupr1 is expressed in various non-small cell lung cancer (NSCLC) cell lines and clinical lung cancer samples. shRNA-mediated knocking down Nupr1 induces severe autophagy disorder. Through Co-immunoprecipitation and mass spectrum, we found that Nupr1 can bind lysosomal-associated membrane protein-2A (LAMP-2A), which suggests that Nupr1 regulates autophagy through interacting with LAMP-2A. However, the exact function of Nupr1 mediating autophagy pathway is unknown. In this grant proposal, the regulatory mechanism of autophagy by Nupr1 will be investigated, including investigating the molecular mechanism of the interaction between Nupr1 and LAMP-2A, exploring regulatory mechanism of autophagy by Nupr1 through binding transcription factors. We will also test the effects of cell functions especially by knocking down Nupr1 in NSCLC cells in vitro and in vivo. Furthermore, the relationship between the expression level of Nupr1 and tumor properties in animals as well as in clinical samples will also be explored. Based on our findings we may find out the regulatory mechanism of autophagy mediated by Nupr1 and its binding partners in NSCLC cells. This study will provide a theoretical basis for developing new therapeutic treatment of NSCLC.

作为肿瘤治疗的靶点，细胞自噬的调控途径是当前研究的热点之一。我们前期的研究表明，非小细胞肺癌细胞系及临床样本中转录调控因子Nupr1高表达，下调Nupr1引起细胞自噬严重失调；利用Co-IP及蛋白质谱检测鉴定了Nupr1与溶酶体受体蛋白LAMP-2A相互作用，提示这种相互作用参与调控非小细胞肺癌细胞自噬途径，但Nupr1如何行使功能的分子机制未知。本课题将继续深入探讨Nupr1调控非小细胞肺癌细胞自噬途径的机制，包括Nupr1与LAMP-2A相互作用的分子机制；探索Nupr1通过结合细胞核内的转录因子调控细胞自噬的机制。进而研究在改变Nupr1表达水平（knockdown）后肿瘤细胞在体外、体内生理特性的变化；再结合动物实验和肺癌临床样本分析，找出Nupr1表达水平与肿瘤特性的关系，为发展针对Nupr1蛋白的非小细胞肺癌治疗新途径提供实验基础。

（一）研究背景.在肿瘤发展的晚期阶段，自噬可以通过缓解肿瘤细胞遭遇的外界压力来促进肿瘤发展的进程。在肿瘤发生发展的过程中，自噬的调控途径是当前研究的热点之一。.我们前期的研究表明，非小细胞肺癌细胞系及临床样本中转录调节因子Nupr1高表达，下调Nupr1引起细胞发生以囊泡化为代表性现象的自噬严重失调，且细胞的增殖变慢，提示Nupr1参与调控非小细胞肺癌细胞自噬途径，但Nupr1行使功能的分子机制未知。.（二）主要研究内容.1. 检测不同分期的非小细胞肺癌组织样本中Nupr1的表达情况，分析Nupr1与肺癌表型的关系。.2. 研究非小细胞肺癌细胞中，Nupr1调控细胞自噬的分子机制：通过RNA-seq的方法找到Nupr1调控的与自噬相关的蛋白，阐明其调控自噬的机制。.3. 研究Nupr1对非小细胞肺癌细胞体外成克隆和体内成瘤能力的影响，阐明其通过自噬影响肺癌表型的机制。.（三）重要结果及其科学意义.1. 证实了Nupr1的表达与肺癌患者的低生存期相关。为肺癌的分子诊断提供了新基础。.2. 揭示了Nupr1通过调控SNAP25来调控自噬溶酶体的外排过程，并且这个过程的失调与细胞囊泡化、体外早熟性衰老以及体内成瘤的抑制密切相关。证实了Nupr1通过调控自噬溶酶体的代谢过程参与肿瘤的发展进程。