EPB49失调在结直肠癌侵袭转移中的作用和调控机制

Previously we identified EPB49 as a metastasis associated suppressor gene in colorectal cancer using gene expression profile. However, the role and potential mechanism of EBP49 in development and progression of human cancer remains to be elucidated. Gene set enrichment analysis (GSEA) uysing public microarray data revealed that EPB49 levels negatively associated with multiple signaling activated gene signatures. Our previous studies have revealed that EPB49 was downregulated colorectal cancer tissues. In addition, the downregulation of EPB49 in colorectal cancer cell lines was associated with promoter hypermethylation. Further experiments showed that silencing of EPB49 could promote the growth, migration and invasion ability of colorectal cancer cells. Moreover, overexpression of EPB49 inhibited the acitivities of Rac1 and RhoA. In the present study, we will establish colorectal cancer cell lines with stable overexpressiing or stable silencing of EPB49. Then we will observe the role of EPB49 deregulation in EMT, cell motility, invasion and metastasis of colorectal cancer, through in vitro methods and orthotopic mouse metastatic models of colorectal cancer. Next, we will analyze the underlying mechanisms through which EPB49 could modulate signaling pathways such as Rac1 and RhoA, using co-immunoprecipitation and GST-pull down assays. We will also analyze other signaing pathways that are essential in modulation of invasion and metastasis though gene exression microarray. At last, we will measure the methylation status in the promoter of EPB49 gene in the colorectal cancer, using MSP and BSP methods. The clinicopathologic significance of methylation status will be analyzed. The results of this project will provide a comprehensive understanding of the role and mechanism of EPB49 deregulation in invasion and metastasis of colorectal cancer.

EPB49基因是我们前期筛选到的结直肠癌转移抑制相关分子，其在肿瘤中的功能和作用机制不清楚。基因富集分析提示，EPB49在结直肠癌中可能影响多条信号通路；预实验结果证明EPB49在结直肠癌组织中表达下调，并与启动子甲基化有关，EPB49沉默能增强结直肠癌细胞的生长迁移能力，EPB49过表达抑制Rac1和RhoA的活性。本项目拟在前期研究基础上，以稳定过表达和干扰EPB49的结直肠癌细胞株为模型，通过体外功能学实验及结直肠癌原位移植动物模型研究EPB49对EMT、细胞运动及结直肠癌细胞侵袭转移能力的影响；采用免疫共沉淀、GST-pull down等技术探讨EPB49调控Rac1、RhoA等信号通路的分子机制；利用表达谱芯片分析EPB49可能调控的其他关键信号通路；采用MSP及BSP法探讨EPB49在结直肠癌中的甲基化水平及临床意义，揭示EPB49在结直肠癌侵袭转移中的信号通路网络和调控机制。

本课题拟检测EPB49在结直肠癌组织中的表达情况，分析其表达与相关临床病理参数之间的关系；探讨EPB49在结直肠癌细胞侵袭和转移过程中的作用和相关的分子机制。我们利用慢病毒建立EPB49稳定过表达和干扰的结直肠癌细胞株，通过Transwel侵袭和迁移实验、划痕实验和三维培养实验和裸鼠原位移植瘤实验检测细胞的侵袭合迁移能力；激光共聚焦技术观察EPB49稳定过表达和干扰后结直肠癌细胞的形态、片状伪足形成和细胞粘附的改变；利用公共数据库（GEO数据库），通过GSEA基因富集的方法分析结直肠癌中EPB49低表达组中上调的信号通路（包括Rac1、RhoA、CDC42等信号通路）；利用westernblot检测EPB49稳定过表达和干扰后Rac1-GTP的表达情况，通过激光共聚焦和免疫共沉淀（Co-Immunoprecipitation, Co-IP）技术验证EPB49和ARHGEF2的相互作用情况以及作用的具体位点，通过恢复实验进一步验证EPB49和ARHGEF2相互作用调控Rac1的活性。本课题得出如下结论：.1..结直肠癌组织中EPB49表达水平显著下调；EPB49的表达水平与分化、Dukes分期、TNM分期、转移和预后等临床病理参数之间呈负相关关系。.2..EPB49过表达抑制结直肠癌细胞的增殖、EMT表型和侵袭迁移能力，而干扰EPB49后则促进肿瘤细胞增殖、EMT表型和侵袭迁移能力；.3..EPB49抑制Rac1信号通路活性，其分子机制可能与EPB49和ARHGEF2相互作用有关；.4..EPB49调节细胞骨架重构、Wnt和MAPK信号通路活性；.5..EPB49基因在结直肠癌中表达下调，其调控机制可能与启动子CpG岛甲基化有关。