甲基化失活基因ENKUR在鼻咽癌中作用机理研究

Several studies showed that epigenetically inactivation of tumor suppressor genes, especially by aberrant promoter methylation, plays a key role in nasopharyngeal carcinoma (NPC) tumorigenesis. Our previous studies showed that high frequency of DNA methylation was possibly occurred in ENKUR promoter, and the mRNA level of this gene was downregulated in NPC. Functional studies found that ectopic expression of ENKUR inhibited cell growth, migration and invasion through regulating cell cycle and EMT pathways. Therefore, we supposed that promoter methylation led to the inactivation of ENKUR gene, which might function as a potential tumor suppressor in NPC. However, little is known about ENKUR in cancer. In this study, we will confirm that promoter methylation leads to ENKUR inactivation in NPC, and clarify the role and mechanism of ENKUR gene as a potential tumor suppressor in NPC tumorigenesis. Based on these results, we will identify the target protein which directly interacts with ENKUR, and the downstream pathways associated with ENKUR and the interaction protein. Finally, we will examine the expression of ENKUR and the interaction protein in NPC tissues, and explore the correlations between ENKUR expression and prognosis and clinical stage. Our study will firstly identify ENKUR as a potential tumor suppressor in NPC, which will provide new understandings about NPC tumorigenesis.

研究表明，启动子甲基化致抑癌基因失活在鼻咽癌发病机制中扮演重要角色。前期研究中我们发现ENKUR启动子在鼻咽癌中可能发生高频率甲基化，并证实其mRNA在鼻咽癌中表达显著下调。功能实验表明，ENKUR通过抑制细胞周期和上皮间质转化抑制鼻咽癌细胞生长、游走和侵袭。据此，我们推测ENKUR在鼻咽癌中为候选抑癌基因，启动子甲基化致其表达失活。迄今为止，尚无ENKUR在肿瘤中的任何报道。本研究拟从如下方面开展：1）证实ENKUR启动子甲基化是其在鼻咽癌中失活的重要原因；2）阐明ENKUR在鼻咽癌中的抑癌基因作用及分子机制；3）鉴定ENKUR的相互作用蛋白，并确定ENKUR通过该蛋白靶向信号通路抑制鼻咽癌生长、游走、侵袭和转移；4）明确ENKUR和该蛋白在鼻咽癌中表达及与临床进展和预后关系。本研究将首次揭示ENKUR为鼻咽癌候选抑癌基因及其作用机制，为深入理解鼻咽癌发病机理提供理论依据。

鼻咽癌是一种发生在鼻咽粘膜的恶性肿瘤，由于广东省发病率位居全国榜首，故又称为“广东癌”。其恶性程度较高，临床转移性极强。蟾蜍毒素目前广泛应用于临床治疗，是一种安全有效的抗癌药物。由于鼻咽癌肿瘤的发生发展涉及多个基因与信号通路的相互作用，以及癌基因的激活和抑癌基因的失活，因此探索与鼻咽癌发生、发展的相关基因及分子信号通路可以有助于揭示鼻咽癌的发病机制。.ENKUR（又名Enkurin）相对分子量为29kDa，由10p12.1染色体上的ENKUR基因编码生成，由8个外显子组成，长3378bp，位于反义链上。2004年研究精子中TRPC（瞬时型感受器电位 ）通道活性和Ca2 +偶联的机制时，通过酵母双杂交筛选发现了ENKUR，其高水平表达于睾丸和犁鼻器，并在其它组织中较低水平。目前ENKUR在肺癌和大肠癌有过报道，但是在鼻咽癌中未见相关报道。. 研究表明启动子甲基化致抑癌基因失活在鼻咽癌发病机制中扮演重要角色。前期研究中 .我们发现ENKUR启动子在鼻咽癌中可能发生高频率甲基化，并证实其mRNA在鼻咽癌中表达显著下调。然后我们构建了稳定过表达ENKUR的5-8F和HONE-1细胞株后通过MTT,EDU,裸鼠皮下成瘤实验,Boyden,划痕实验及裸鼠肺转移模型实验证明ENKUR在鼻咽癌中发挥抑癌作用。机理研究表明，ENKUR通过与β-catenin相互作用并阻碍核β-catenin的积累来抑制β-catenin诱导的肿瘤信号和活性。最后，我们观察到安全有效的抗癌药物蟾蜍毒素（CB）通过抑制PI3K/AKT/C-Jun上调了ENKUR的表达水平，从而增强了ENKUR介导的NPC细胞的体外体内增殖和转移。我们的实验结果阐明了ENKUR这一未知基因的抑癌作用，并说明了蟾蜍毒素对ENKUR的调控机理，对鼻咽癌的治疗有指导意义。