转录因子FOXK1通过CTGF和KLF2调控胃癌细胞侵袭和转移

Transcriptional factor FOXK1 is a member of the FOX family, involved in the cell growth and metabolism. The disorder expression of FOXK1 leads to a variety of diseases and may play an important role in the development of tumor. In our preliminary data, high expression of FOXK1 was detected in gastric cancer (GC) compared with normal tissue. FOXK1 also induced the proliferation and invasion and promoted Epithelial-Mesenchymal Transition (EMT) in GC cells. Using cDNA-microarray, we found knockdown of FOXK1 inhibited expression of CTGF and enhanced expression of KLF2, which may be related to the invasion and metastasis of GC. ChIP assay verified transcriptional factor FOXK1 bound with the promoter of CTGF and KLF2. Based on these experiments, we hypothesis that FOXK1 induce the proliferation and migration of GC cells through transcriptional regulation of CTGF and KLF2. In this proposal, we are planning to investigate the role of FOXK1 on proliferation, EMT, invasion and metastasis in GC, assess the transcriptional regulation of FOXK1 to promote CTGF expression and inhibit KLF2 expression individually. Furthermore, subcutaneous and orthotopic xenograft model in nude mice will be established to confirm the role of FOXK1, CTGF and KLF2 during GC progression in vivo. Statistical analysis will demonstrate the relationship between expression of FOXK1, CTGF and KLF2 and prognosis of GC patients. .In view of previous studies and proposal designed above, this study would elucidate for the first time that FOXK1 acts as an oncogene in GC and plays an important role in GC proliferation, invasion and metastasis. Moreover, we propose to demonstrate the interaction between FOXK1 and CTGF or KLF2 individually, yielding the mechanisms of FOXK1 induced invasion and metastasis. This study would provide a novel opinion and method for molecular targeted treatment and prognosis of GC.

FOXK1转录因子属于Fox家族中的一员，其表达异常可引起多种疾病，并可能在肿瘤发生发展中起到重要作用。预实验发现，FOXK1在胃癌组织中表达比正常组织高，促进胃癌细胞增殖和侵袭，引起上皮-间质（EMT）转化。通过siRNA沉默FOXK1，利用基因芯片筛选出差异表达基因CTGF和KLF2，与胃癌侵袭转移相关；通过染色质共沉淀实验(Chip)，确定转录因子FOXK1对CTGF及KLF2启动子区存在转录调控作用。因此，我们提出科研假设：FOXK1能够促进胃癌增殖和EMT，该作用可能是增强CTGF，抑制KLF2的表达调控胃癌细胞侵袭和迁移。本项目拟探讨FOXK1在胃癌增殖、EMT和侵袭转移中的调控作用；评估FOXK1对CTGF分子表达的促进作用；明确FOXK1通过抑制KLF2影响胃癌细胞的侵袭和转移；体内验证上述作用与机制；统计分析三个基因的表达和病人预后的关系。完成后具有较大的理论与临床价値。

背景和目的: c-Jun、Snail、FOXK1和Vimentin是致癌基因，本研究旨在明确c-Jun/FOXK1、 Vimentin/FOXK和Snail/FOXK信号轴调控胃肠癌细胞生物行为的分子机制。.方法：通过构建c-Jun、Vimentin、Snail与FOXK1稳定表达株或siRNA干扰、western blot、qPCR、Coimmunoprecipitation (Co-IP)、Chromatin immunoprecipitation (ChIP)、Luciferase 活性、Confocal microscopy、明胶酶谱以及构建小鼠模型等方法开展实验。.结果：1. FOXK1在胃癌组织中的表达要高于癌旁正常组织；过表达FOXK1促进胃癌细胞的增殖侵袭与转移能力；FOXK1参与了TGF-β1诱导的胃癌细胞EMT。c-Jun能与FOXK1的启动子区域结合并启动下游转录；FOXK1与c-Jun的表达有关联且与胃癌患者的预后负相关。在体外体内模型中，敲减c-jun能降低FOXK1对胃癌细胞增殖、转移的促进作用。2. FOXK1 和Vimentin相互作用及在胃癌细胞中表达呈正相关；两个分子共表达与胃癌的预后负相关；下调Vimentin 抑制FOXK1 诱导的胃癌EMT；Vimentin-siRNA 影响FOXK1 诱导的胃癌细胞形态；FOXK1 与Vimentin 在胃癌细胞的侵袭和转移中有协同作用。3. Snail与FOXK1在大肠癌组织同向表达并具有相关性；FOXK1启动子区域有Snail结合位点；Snail-FOXK1信号轴诱导大肠癌细胞EMT及侵袭能力；Snail与FOXK1协同促进大肠癌的区域淋巴结转移；FOXK1和Snail的表达在大肠癌组织中存在相关性；FOXK1和Snail的表达与大肠癌患者预后负相关；在小鼠模型中，Snail和FOXK1协同促进大肠癌的侵袭转移。.结论：FOXK1和c-jun、Vimentin及Snail的相互作用，在胃肠癌增殖侵袭转移中扮演重要的角色。