E3泛素连接酶TRIM7调控NF-κB在肺癌中的功能及其分子机制研究

E3 ubiquitin ligase is an important therapeutic target in lung cancer for their ability to regulate proteins stability and functions. Extensive study on the molecular mechanism by which E3 ligases regulate cellular processes would lead to higher therapeutic effect and lower side-effect of lung cancer. TRIM7 is a member of tripartite motif family (TRIM). In previous study, the longest isoform of TRIM7 (GNIP) has been reported to bind to RACO-1 and promote lung cancer through its E3 ubiquitin ligase function. Recently,however,we found that the shortest isoform of TRIM7 (TRIM7s) inhibited the growth of lung cancer cells, which means TRIM7 showing distinct functions between different isoforms in the regulation of lung cancer. Furthermore, TRIM7s inhibited the protein expression of NF-κB without an effect on its mRNA level, and there was a interaction between TRIM7s and NF-κB subunit p65. In our present study, we investigate the mechanisms underlying the regulation of NF-κB signaling pathway by TRIM7s through Co-IP, GST-pull down, Mass, and animal lung cancer model experiments. Our study indicates the two distinct functions of TRIM7 and suggests the function of E3 ubiquitin ligase in the development of lung cancer, which paves the way for novel treatment strategies for lung cancer.

E3泛素连接酶由于其泛素化修饰功能成为肺癌治疗中的重要靶点，而阐明其发挥功能的分子机制对于提高肺癌治疗特异性并降低副作用具有积极意义。TRIM7为三结构域蛋白家族成员，此前报道显示其最长亚型GNIP1作为E3连接酶与RACO-1蛋白结合从而促进肺癌的发展。但是，本课题组最新研究表明，其同样具有RING结构域的最短亚型TRIM7s与GNIP1在肺癌中的功能迥异，可显著地抑制肺癌细胞增殖。后续研究发现，TRIM7s抑制NF-κB的蛋白表达而不影响其mRNA水平，且二者之间存在蛋白相互结合。因此，本项目中，我们将采用免疫共沉淀、GST-pull down、质谱、裸鼠肺癌模型等技术从体内外进一步揭示TRIM7s对NF-κB调控的分子机制及其在肺癌中的功能，阐明TRIM7在肺癌中的双重作用机理。本项目的实施有助于明确E3泛素连接酶在肺癌中的不同作用及其调控过程，从而为特异性靶向治疗肺癌提供新思路。

trim7（tripartite motif-containing 7）基因编码了GNIP1，GNIP2，GNIP3和TRIM7等至少4个亚型的蛋白质。其中，氨基酸序列最长的亚型GNIP1已经被报道在肺癌中发挥着原癌基因的作用。TRIM7（在本项目申请书中我们为了区别于trim7基因故写作TRIM7s）是现在已知四个亚型中氨基酸序列最短的蛋白，其在羧基端（C末端）有15个碱基与GNIP1不同。然而有趣的是，我们的研究显示TRIM7s在肺癌中的功能与GNIP1截然相反。与癌旁组织相比，TRIM7s在肺癌组织中是低表达的，且与肺癌的临床分级负相关。此外，肿瘤异种移植实验也证实TRIM7s能够抑制小鼠皮下肿瘤的生长。在细胞中，TRIM7s能够显著地抑制肺癌细胞系的增殖和迁移，同时促进癌细胞的凋亡。进一步研究发现，TRIM7s能与NF-kappaB信号通路中的p65蛋白相互作用并泛素化修饰p65蛋白，通过TRIM7s羧基末端的15个氨基酸与p65蛋白相互结合并添加泛素分子，导致p65蛋白经泛素化依赖性的26S蛋白酶体降解，最终抑制NF-kappaB信号通路的转导。这些研究揭示了一个新的由TRIM7s参与的NF-kappaB信号通路负调控机制，并证实TRIM7s在肺癌中发挥了肿瘤抑制因子的功能。