AJUBA调控IFIT2介导的细胞凋亡及其在结直肠癌发展中的作用与机制研究

The LIM protein AJUBA functions as scaffold, which participates in the assembly of multiple protein complexes and involves in different kinds of biological processes such as mitosis, gene transcription, cell adhesion, differentiation, proliferation and migration. In this study, we found that the expression of AJUBA was positive correlated with the malignancy of colorectal cancer cell lines. Knockdown of AJUBA results in the apoptosis of colorectal cancer cell. And also, AJUBA was highly expressed in the clinical specimens of colorectal cancer. Therefore, we proposed that AJUBA had vital roles in colorectal cancer development. In order to demonstrate the mechanism of AJUBA in the progression of colorectal cancer, we use gene expression profile analysis to explore the potential target proteins of AJUBA in colorectal cancer by comparing AJUBA knockdown and normal cells. We found that knockdown of AJUBA had significantly effects on almost all of the Interferon-Stimulated (ISG) genes, including the apoptosis-inducing protein IFIT2 which was regulated by JAK/STAT signaling. The higher expression of IFIT2 in AJUBA knockdown cell was confirmed by real-time PCR and western blotting. Interestingly, we found that AJUBA could block STAT1 phosphorylation and translocation from cytoplasm into nucleus. Thus, this project would focus on demonstrating the roles of AJUBA in the progression of colorectal cancer. In this project, 1) we would illuminate if IFIT2 is the potential target of AJUBA in regulating cell apoptosis and colorectal cancer development. 2) we would define if AJUBA regulating IFIT2 expression through JAK/STAT signaling pathway. 3) we would explore if AJUBA could be used as diagnostic marker for colorectal cancer by analysis the clinical specimens. In conclusion, this project presented here would hopefully illuminate the functions and molecular mechanism of AJUBA in regulating cell apoptosis and colorectal cancer development. This study is likely to highlight a new path to develop anti-tumor drugs for colorectal cancer.

LIM蛋白AJUBA参与调控基因转录、细胞分化、增殖、迁移与粘附等多种生物学功能。本项目发现AJUBA表达水平与结肠癌细胞的恶性程度呈正相关，且AJUBA在临床结直肠癌组织中高表达。可见AJUBA与结肠癌的关系密不可分。为深入研究其作用机制，我们对AJUBA敲低的结肠癌细胞系进行表达谱分析，发现一系列干扰素诱导基因的表达受到影响，其中已证实促凋亡蛋白IFIT2的上调。IFIT2是JAK-STAT通路中的下游靶基因。我们初步发现AJUBA可抑制STAT1的磷酸化及入核。我们将展开如下研究：1)明确IFIT2是AJUBA抗凋亡和促进肿瘤发展的下游分子。2)阐明AJUBA是否通过JAK/STAT信号调控IFIT2及分子机制。3)分析临床样本中IFIT2与AJUBA的相关性，探寻新的结直肠癌诊断标志物及治疗靶点。期望通过研究AJUBA调控结直肠癌发展的下游分子机制，为其临床治疗提供新的理论基础。

LIM蛋白AJUBA参与调控基因转录、分化、增殖、迁移与粘附等多种生物学功能。AJUBA作为一个细胞内的“锚定蛋白”，与细胞内的多种蛋白结合并参与多条信号通路的调节。研究发现AJUBA蛋白在非小细胞肺癌，恶性间皮瘤，前列腺癌和肝癌等多个肿瘤中具有抑癌基因的功能，但在乳腺癌，食管鳞癌等肿瘤中AJUBA具有促癌的作用，以上这些研究表明AJUBA在肿瘤中具有两面性。我们前期利用IHC染色结直肠癌标本发现AJUBA高表达，使用TCGA数据库发现在864例结直肠癌组织中AJUBA高表达。我们提出科学问题：AJUBA在结直肠癌中高表达有什么作用？进一步探讨AJUBA为什么在肿瘤中具有两面性？我们的研究首先发现了AJUBA在结直肠癌中高表达，分别利用结直肠癌细胞株SW1116和Caco-2证实AJUBA过表达可以促进增殖，敲除可以抑制增殖，利用体外裸鼠成瘤实验也证实AJUBA过表达可以促进成瘤。进一步研究发现AJUBA过表达可以抑制结直肠癌细胞的凋亡，利用RNA-Seq研究发现干扰素介导的JAK-STAT信号通路参与调控。主要机制是AJUBA竞争性的阻断JAK1与其受体IFNGR1的结合，抑制了STAT1的磷酸化及入核，降低了下游IFIT2的表达，阻止了结直肠癌细胞凋亡，促进了结直肠癌的发生。进一步通过利用蛋白质免疫共沉淀等一系列技术我们找到了AJUBA竞争性的阻断JAK1与其受体IFNGR1的结合的位点，根据这段序列开发了小分子多肽EPSGEPSG，初步发现可以抑制结直肠癌的发生，为后续的成药奠定基础。AJUBA在结直肠癌中高表达的原因，我们研发发现BMP-Smad1信号通路中的Smad1通过上调了AJUBA的表达，促进了EMT的发生，导致了结直肠癌的转移。进一步在胰腺癌中我们也发现了AJUBA的高表达，主要机制是AJUBA与SP1形成复合体促进了SP1的高表达，促进了胰腺癌的发生和发展。我们的研究意义在阐明了AJUBA作为癌基因在结直肠癌和胰腺癌中的重要作用，为其诊断和治疗提供了靶标。开发的抗肿瘤的小分子多肽在也是我们将要研究的重点。