Ovol2调节结肠癌侵袭转移的机制研究

Ovol2 is a member of a family of zinc finger transcriptional repressors. Our published studies demonstrated that Ovol2 plays a pivotal role in development; however, weather Ovol2 regulates the formation and progression of cancers is still unknown. Our previous study of this proposal showed that Ovol2 may inhibit Wnt signaling by competition with ?-catenin for binding to TCF4 and regulate nucleus-membrane distribution of ?-catenin. On the other hand, Ovol2 was also found to be a positive target of Wnt signaling. Hence a negative feedback loop between Wnt signaling and Ovol2 exists in the colon cancer cells, which may keep the Wnt signaling under a threshold level to inhibit cancer invasion and metastasis. Our study also showed that Ovol2 loses its response to Wnt signaling though the methylation of its promoter region along with the progression of colon cancer, which results in an impaired repression ability to Wnt signaling. Consequently, the output of Wnt signaling is higher than the threshold in these cells which leads to the induction of EMT process. In addition, Ovol2 was found to inhibit Notch signaling though repression of the expression of Notch1, a prominent Notch receptor. Therefore, loss of function of Ovol2 during cancer progression may also induce the intravasation process though inhibition of Notch signaling. In this proposal, we will systematically study the mechanism and function of Ovol2 in regulating the invasion and metastasis of colon cancer, in molecular, cellular, transgenic /knockout mouse tumor models and clinical tumor sample levels. Our study will reveal a possibility that Ovol2 may act as a novel target for diagnosis and treatment of invasion and metastasis of colon cancer.

Ovol2是含锌指蛋白结构的转录抑制因子，我们之前的研究发现，Ovol2在组织发育中发挥重要作用，然而在肿瘤发生发展过程中的作用仍然未知。本课题前期实验发现， Ovol2在初期结肠癌细胞内抑制Wnt信号，同时Ovol2又是Wnt信号的正向靶基因。二者组成一个负反馈环使Wnt信号输出维持在一定水平，抑制肿瘤侵袭转移；我们还发现，随着结肠癌的进展，Ovol2启动子区发生甲基化，失去对于Wnt 信号的反应，导致其表达降低或消失，解除对于Wnt信号的抑制作用。因而，Wnt信号输出高于门槛值而启动EMT。另外，Ovol2通过抑制Notch1表达而抑制Notch信号，因此进展期Ovol2失活将通过Notch信号促进肿瘤Intravasation 渗入血管。本课题将从分子、细胞、常位肿瘤模型以及基因修饰小鼠肿瘤模型并结合临床标本分析，系统研究Ovol2在结肠癌侵袭转移发生中的作用及其相关的分子机理。

Ovol-2是进化上相当保守的一种转录因子，它与胚胎发育之间的关系已经被很多研究所报道，但是Ovol-2与肿瘤的发生发展关系还知之甚少。本文主要从Ovol-2是如何调控结直肠癌的转移与侵袭方面做了详细的研究。.首先我们从结直肠癌病人样本的组织芯片中发现肿瘤中Ovol-2含量低的病人预后生存期更短。然后我们通过体外细胞增殖实验、裸鼠皮下成瘤实验、小鼠免疫组化实验、HE染色实验证实了Ovol-2不能够抑制结直肠癌的增殖。于是我们猜测Ovol-2可能是通过抑制结直肠癌转移和侵袭过程，延长患者的寿命。于是我们通过细胞划痕实验、Transwell实验、裸鼠尾静脉肿瘤细胞注射实验、裸鼠原位肿瘤细胞注射实验、HE染色实验、免疫荧光实验证实了Ovol-2能够抑制结直肠癌的转移。.接下来我们通过Oncomine数据库筛查与肿瘤转移有关与Ovol-2共表达的基因，通过免疫组化实验、细胞免疫荧光实验、蛋白印迹实验我们证实了Ovol-2能够抑制结直肠癌细胞的EMT进程。我们通过qPCR array找到了与Ovol-2相关性较高的关键蛋白SLUG。并通过免疫印迹实验、荧光实时定量PCR实验、细胞划痕实验、Transwell实验、荧光素酶报告质粒活性检测实验证实了Ovol-2是通过抑制SLUG来抑制结直肠癌的EMT进程。我们通过点突变SLUG启动子上的Ovol-2结合位点发现Ovol-2并不是直接负调控SLUG的表达，而是通过抑制Wnt信号通路的转录活性负调控SLUG的表达。.我们通过荧光素酶报告基因活性检测实验、免疫染色质共沉淀实验、免疫组化实验发现Ovol-2是Wnt信号通路的直接靶基因。由此我们提出Ovol-2与Wnt信号通路之间存在的负反馈作用在结直肠癌的转移与侵袭中具有重要的调控作用。.