Twist相关蛋白（FOS、RBL2）在胃腺癌演进中的调控作用和机制研究

Twist, a key transcription factor during embryonic development, has been recently shown to play an important role in enhancing tumor invasion in several types of human cancer via regulating epithelial mesenchymal transition, but the mechanisms are unclear. In our previous studies, we have shown that depletion of Twist leads to changes of gastric cancer cell morphology and reduced ability of invasion. In addition, the protein levels of FOS and RBL2 change remarkably upon suppression of Twist expression in multiple gastric cancer cell lines,which were published in 2009. To test the roles of FOS and RBL2 in Twist -dependent cancer development and clarify the relationship among these proteins, we will design vectors to overexpress or shRNA vectors to suppress the expression of these genes to test their roles on cell proliferation and invasion ability in multiple gastric cancer cell lines. Furthermore, we will design vectors to manipulate the expression of p53 gene and others such as JUN, JUNB and JUND to determine 1) whether the roles of FOS and RBL2 are dependent on functional p53, and 2) whether the effects of FOS can be antagonized by other proteins. Finally, we will test the roles of FOS and RBL2 on cells' response and sensitivity to chemotherapy drugs and irradiation. Completion of this proposal will help to identify unknown target genes regulated by Twist, to improve understanding the mechanisms for Twist-associated increased cell proliferation and enhanced cell invasion, and to provide experimental basis for designing molecular targets for gastric cancer therapy in the future.

Twist蛋白作为转录因子，通过调控上皮间质转化而促进肿瘤细胞的转移，其确切机制尚待研究。我们前期研究内容作为发表于2009年：下调表达Twist基因可导致高转移潜能胃腺癌细胞侵袭能力的下降；通过基因芯片筛选出Twist基因沉默前后差异表达显著的两个基因：FOS和RBL2。结合系列前期研究结果，我们推测：Twist通过上调FOS基因表达、下调RBL2基因表达，提高胃癌细胞增殖能力、抑制细胞僵死过程，进而促进胃腺癌细胞的演进和转移。因此，本项目将在侵袭能力不同的胃腺癌细胞系中，验证FOS和RBL2基因是否为Twist靶基因，进而研究其发挥作用的可能的通路机制。本项目的开展，有利于Twist蛋白潜在靶基因的发现与深入研究；有助于阐明胃腺癌转移的分子机制、发现对胃腺癌临床治疗有意义的治疗靶点。

参照项目申请书的研究内容，本课题首先使用使用Real Time-PCR和Western blot的方法对人胃癌细胞中TWIST蛋白、Fos和Rbl-2基因表达进行了检测，我们发现，TWIST在肿瘤细胞中的蛋白含量明显高于GES-1细胞，Fos基因mRNA在肿瘤中的含量明显高于GES-1细胞，Rbl-2基因mRNA在肿瘤中的含量则明显低于GES-1细胞；进一步的通过CHIP-PCR实验发现Twist与Fos基因启动子的结合明显增强，而与Rbl-2基因启动子的结合明显减弱。而且，Fos基因沉默或者Rbl-2基因过表达可以逆转Twist基因过表达对细胞增殖和迁移的影响， Fos基因过表达或者Rbl-2基因沉默可以逆转Twist基因沉默对细胞增殖和迁移的影响。通过以上研究，我们确定Twist参与胃腺癌转移的机制之一为通过增强下游基因Fos的表达和抑制下游基因Rbl-2的表达来增强肿瘤细胞的增殖和迁移能力。在此基础上，本课题还进行了拓展研究，本研究还发现肿瘤组织中除了Twist以外，Apelin的高表达也与肿瘤分化程度、淋巴结转移和远处转移密切相关。我们还发现miR-367与Twist在胃腺癌组织样本中的表达有显著负相关，并通过luciferase assay证实miR-367能够与TWIST 3’-非翻译区结合并抑制其mRNA的翻译。过表达实验也进一步证实了miR-367调控Twist表达，进而抑制胃腺癌转移这一机制。项目资助发表SCI研究论文1篇，另有1篇已接收。培养博士生1名，硕士生2名，其中1名已经取得硕士学位，2名在读。项目投入经费23万元，支出17.9496万元，各项支出与预算相符。剩余经费5.0504万元，剩余经费计划用于本项目研究后续支出。