FMNL3基因在结直肠癌上皮-间质转化中的作用及调控机制

FMNL3 is a new found member of diaphanous-related formins (DRFS). In our previous study, we found FMNL3 gene is involved in epithelial-mesenchymal transition (EMT) maintainance in human colorectal carcinoma (CRC). In addition, we raised a hypothetical EMT signaling pathway net of FMNL3 invovled in. In this study, we will verification this hypothesis. Firstly, we will observe the expression of FMNL3 and EMT marker in CRC tissues and cell lines, and analysis the relationship with each other. Moreover, we will investigate the effects of FMNL3 transfection or knockdown on EMT marker, metastasis in vivo and invasion in vitro in CRC cells. Thirdly, we will detect the change of cell morphology, the expression of EMT marker and key transcription factor after treating FMNL3 overpressed or knockdown CRC cells with exogenous TGF-β. Fourthly, we will inspect the influence of the protein expression of EMT signaling pathway such as Wnt/β-catenin、PI(3)K/AKT、Ras/MAPK pathway, the influence of the activity of the kinase and the key transcription factor by FMNL3 transfection or knockdown. Lastly, we will observe the effects on these three signaling pathways by treating FMNL3 transfected or knockdown CRC cells with the suppressors of GSK3βor/and PI(3)K/AKT or /and MAPK. Our study aims not only to illustrate molecular mechanism of FMNL3 in the regulation of EMT and its signaling pathway, but also to reveal the new relationship of FMNL3 gene with CRC metasasis.

FMNL3是新近发现的一个DRFs家族成员。前期研究中我们发现FMNL3与结直肠癌EMT相关，并提出FMNL3参与结直肠癌EMT的可能信号通路网络。本研究将验证此设想，首先观察分析结直肠癌组织和细胞中FMNL3与EMT标志物表达的关系；接着观察FMNL3转染/干扰对结直肠癌细胞EMT标志物及其体内外侵袭转移能力的影响；然后用外源性TGF-β刺激FMNL3转染/干扰细胞，观察细胞形态、EMT标志物及关键转录因子的表达改变；再检测FMNL3转染/干扰对EMT相关的Wnt/β-catenin、PI(3)K/AKT、Ras/MAPK信号通路蛋白的表达、激酶和转录因子活性的影响；最后分别或联合给予GSK3β、PI(3)K/AKT、MAPK等抑制剂处理，观察其对以上三种信号通路的影响。旨在阐明FMNL3参与结直肠癌EMT的分子机制，揭示FMNL3参与结直肠癌转移调控的新联系。

FMNL3是新近发现的一个DRFs家族成员。前期研究中我们发现FMNL3与结直肠癌EMT相关，并提出FMNL3参与结直肠癌EMT的可能信号通路网络。本研究将验证此设想，首先观察分析结直肠癌组织和细胞中FMNL3与EMT标志物表达的关系；接着观察FMNL3转染/干扰对结直肠癌细胞EMT标志物及其体内外侵袭转移能力的影响；然后用外源性TGF-β刺激FMNL3转染/干扰细胞，观察细胞形态、EMT标志物及关键转录因子的表达改变；再检测FMNL3转染/干扰对EMT相关的Wnt/β-catenin、PI(3)K/AKT、Ras/MAPK信号通路蛋白的表达、激酶和转录因子活性的影响；最后分别或联合给予GSK3β、PI(3)K/AKT、MAPK等抑制剂处理，观察其对以上三种信号通路的影响。.综合以上研究结果，我们发现: 1、FMNL3 在结直肠癌组织和细胞株中的表达与 EMT 上皮标志物的表达呈负相关，而与间质标志物呈正相关；2、FMNL3能显著上调结直肠癌细胞EMT间质标志物的表达并下调上皮标志物的表达，并促进结直肠癌细胞体外侵袭和迁移；3、FMNL3能显著促进结直肠癌细胞经TGF-β诱导的间质表型的形成及其转录因子snail、twist的表达；4、FMNL3能显著促进结直肠癌细胞PI(3)K/AKT信号通路中p-PI3K、p-AKT的表达及Ras/MAPK信号通路蛋白中p-MAPK、p-MEK的表达，而对Wnt/β-catenin通路中p-GSK3β蛋白表达无影响；并上调PI(3)K、AKT、MAPK等激酶的活性及snail、twist的转录活性。因此，FMNL3通过PI3K/AKT通路及Ras/MAPK通路促进结直肠癌细胞发生EMT。.在国家基金委的资助下，本课题全部计划内容已经完成，目前共发表文章5篇，其中SCI论文1篇，中文核心期刊4篇；余下部分内容正待发表。