肺癌细胞S100A16核转位调控MRPL14表达维系氧化应激条件下线粒体蛋白质稳态的作用机制研究

Oxidative stress plays a "double-edged sword" role in the course of lung cancer. In the process of metastasis, cancer cells need to resist oxidative stress in order to survive in the target organs. However, the specific mechanism of metastasis is still unclear. We previously have found that over-expression of S100A16 can maintain the stability of mitochondrial membrane during brain metastasis of small cell lung cancer, thus contributing to its survival in the brain. Further studies showed that S100A16 could translocate into the nucleus under oxidative stress and maintain the stability of mitochondrial membrane in lung cancer cells. Nuclear translocation of S100A16 can up-regulate the expression of mitochondrial ribosomal protein MRPL14. Functional analysis of MRPL14 suggests that it is related to the stability of mitochondrial membrane. Based on these foundings, our study is intended to carry out the following work: (1) To explore the molecular mechanism of S100A16 migration into the nucleus under oxidative stress; (2) To determine the molecular mechanism of regulation of MRPL14 expression by translocation of S100A16 into nucleus；(3) To elucidate the role and mechanism of upregulation of MRPL14 to maintain mitochondrial homeostasis under oxidative stress after translocation of S100A16 into nucleus; (4) To analyze the clinical relationship between the expression of S100A16/MRPL14 in lung cancer tissues during the occurrence and metastasis of lung cancer. The completion of this study can provide experimental basis for the study of the mechanism of lung cancer cells resisting mitochondrial oxidative stress to maintain cell survival during metastasis. It is expected to provide a potential intervention target for clinical treatment of lung cancer metastasis.

氧化应激在肺癌病程中发挥着“双刃剑”的作用，在转移过程中癌细胞多需要抵抗氧化应激才能在靶器官生存下来，其具体机制尚不清楚。我们前期工作发现，应激蛋白S100A16可以维系小细胞肺癌细胞线粒体膜的稳定性，帮助其在脑内存活。进一步结果提示氧化应激条件下S100A16由细胞质转位入细胞核，并上调线粒体核糖体蛋白MRPL14表达，维持线粒体膜的稳定。为此，本项目拟开展如下工作：①解析氧化应激条件下S100A16转位入核的分子机制；②探讨转位入核的S100A16调控线粒体核糖体蛋白MRPL14表达的机制；③探明S100A16核移位后上调MRPL14维系氧化应激条件下线粒体功能稳态的作用及机制；④分析肺癌患者组织中S100A16/MRPL14的表达与肺癌发生及转移的临床关系。本项目的完成可望为肺癌细胞抵抗线粒体氧化应激从而维系转移期生存的研究提供实验佐证，并可为肺癌转移防治提供潜在干预靶点。

非小细胞肺癌是肺癌的主要病理类型，恶性程度高，5年总体生存率比较低。氧化应激是肿瘤发生发展过程中的重要影响因素，在肿瘤发生的早期，一定程度的氧化应激可以靶向DNA引起驱动基因的突变，促进肿瘤细胞的持续增殖，诱导肿瘤的发生；另一方面肿瘤细胞表现出很强的抗氧化应激能力，能够维系细胞内ROS的稳定，进而抵抗氧化应激引起的细胞过度损伤和死亡。因此深入探索非小细胞肺癌发生发展过程中氧化应激调控相关的分子机制具有重要的理论和临床意义。S100A16 属于S100 家族成员之一，其结构不同于其他家族成员。已有研究表明S100A16在多种肿瘤组织中高表达，与肿瘤的进展和转移密切相关，但是对于S100A16在非小细胞肺癌中的作用和分子机制知之甚少。本课题通过对公共数据库和临床组织样本的分析发现，S100A16在非小细胞肺癌组织中高表达，且与肺腺癌患者的不良预后相关。体外过表达肺腺癌细胞中S100A16可以明显抵抗氧化应激对细胞的损伤，同时体内实验表明敲低S100A16可以明显抑制肺癌的发展。深入探索发现适量氧化应激可以诱导S100A16发生核转位，利用蛋白质免疫沉淀和质谱分析技术，筛选出介导S100A16发生核转位的结合蛋白-NOLC1。利用免疫共沉淀和激光共聚焦技术证实NOLC1的C端601-700氨基酸区域是与S100A16结合并帮助其转运至细胞核的关键结构域。此外通过生物信息学方法联合分析S100A16敲低转录组数据和TCGA-LUAD队列数据，提示核内S100A16可能通过调控可变剪切过程发挥其作用。进一步分析蛋白质谱数据并利用免疫沉淀技术证实S100A16可以与hnRNPM和MagoHB相互作用形成复合体，生信分析发现该复合体可能通过可变剪切方式调控影响线粒体内核苷酸代谢和嘌呤代谢的相关基因表达，进而发挥其抵抗氧化应激损伤的作用。综上所述，本研究阐明了核内S100A16通过靶向可变剪切过程非小细胞肺癌进展的分子机制，可望为临床非小细胞肺癌的防治提供新的思路和干预的靶点。