EB病毒癌蛋白LMP1通过下调microRNA-203诱导上皮间质转化及其调控机制的研究

Nasopharyngeal carcinoma (NPC) is notorious among head and neck cancers since it is highly associated with Epstein-Barr virus and highly metastatic. Our previous study demonstrated that miR-203 was significantly decreased with a role of EBV-encoded oncoprotein latent membrane protein (LMP1) both in epithelial and NPC cells. It was also showed that the miR-203 downregulation is related to a promoted tumor incidence in a manner depending on the NF-κB pathway. NF-κB and miR-203 are reported to be involved in the invasive and metastatic cancers by facilitating or inhibiting epithelial-mesenchymal transition (EMT) respectively, whereas it is not clear for their relationship. The regulatory mechanism of EMT at the upstream and downstream of miR-203 is largely to be clarified, especially in the EBV-associated NPC. Based on our previous results and the models of EBV-infected epithelial and NPC cells, this project would focus on the regulatory mechanism of EMT induced by EBV-LMP1 via the miR-203 downregulation. The mechanism is to be explored on that NF-κB inhibits the transcription directly or through EMT-related transcription factor indirectly. Whether CHD6, which is potentially related to EMT, is a novel target gene of miR-203 and CDH6 and is also regulated by NF-κB would be studied. The interrelationship among the crucial factors within the whole pathway from LMP1 to EMT would be confirmed both in the cell model study and in clinical samples. The study of this project would provide further evidence for our fully understanding about the EBV-associated etiology during the development of NPC.

与EB病毒高度相关及高度转移性是鼻咽癌（NPC）的重要特征。本项目前期研究发现，EBV通过其癌蛋白LMP1在上皮细胞及NPC中下调miR-203的表达、从而促进肿瘤发生，这种作用与转录因子NF-κB信号通路有关。据报道，NF-κB和miR-203均可通过促进或抑制上皮间质转化（EMT）参与肿瘤的侵袭转移，但是它们之间的关系尤其是在EBV相关性NPC中，miR-203的上下游通路在参与EMT中的调控机制不清楚。本项目以EBV感染的上皮和NPC细胞模型为基础，研究NF-κB是否直接抑制miR-203基因转录或通过其它EMT相关分子下调miR-203及机制，研究miR-203对肿瘤转移相关的靶基因如CDH6的调节及NF-κB是否通过CDH6调节miR-203表达，研究从LMP1到EMT通路中关键分子间的相互关系，并在临床样本中进行检测验证。本研究为EBV在NPC发生中的病因学机制提供新的依据。

EB病毒（EBV）与上皮来源的鼻咽癌（NPC）高度相关，其致瘤机制仍不很清楚。上皮相对特异表达的miR-203是我们发现在鼻咽癌中下调、且被EBV癌蛋白LMP1下调的microRNA，它的下调能促进上皮细胞的增殖。但是，miR-203如何被LMP1下调、下调后是否在NPC发生的其它进程尤其是转移相关关键步骤如EMT等，以前没有被揭示。而这些问题的解决，对于靶向EBV的NPC诊断和治疗很重要。在本项目中，研究了LMP1如何下调miR-203，miR-203下调激活与转移相关的靶基因及其具体功能，化学抑制剂及药物对调控通路的逆转能力，关键分子的表达及外泌对EBV相关性鼻咽癌致病及治疗的意义评估。研究发现，LMP1通过其经典激活的NF-κB，结合到miR-203编码基因启动子区的转录起始位点（TSS）近端，抑制miR-203的表达。同时，LMP1能促进miR-203基因启动子的甲基化，也具有一定的抑制miR-203表达的作用。鉴定了miR-203的新靶基因CDH6在NPC中高表达。钙粘素6（CDH6）是一个在胚胎期活跃、在成体组织局限表达的分子，这种重激活体现了癌症的重要特征。对CDH6的功能研究显示，它能促进鼻咽癌细胞的EMT、迁移和侵袭。骨转移相关转录因子RUNX2是miR-203的另一个靶基因，已被证实是CDH6的激活因子。在本研究中，RUNX2也被激活。可见，miR-203在维持鼻咽正常特性中采取了双重抑制措施。使用NF-κB抑制剂能逆转LMP1/NF-κB/miR-203/CDH6通路的作用与NPC的EMT。这个通路分子的表达关系在EBV阳性的鼻咽癌和裸鼠移植瘤组织中均得到验证。另外，发现一种药物具有促进miR-203外泌的作用，研究在进行中。GEO临床数据分析表明，miR-203下调与NPC复发转移增加显著相关、降低患者的5年生存率。新近报道显示，靶向CDH6在肾癌等的治疗中很有效，这证实了我们的结果，CDH6可望成为NPC治疗靶标。由于RUNX2-CDH6处在TGF-β通路的下游，因此，CDH6也是NF-κB与TGF-β这两条重要通路间对话的节点蛋白。通路间对话也是癌症的特征，而寻找节点蛋白在癌症研究中有重要意义。通过本项目研究，发现了EBV能促进细胞获得癌症趋向的重要特征，更好地了解了EBV的新的肿瘤病因学机制，并找到数个新的NPC诊断治疗的潜在分子靶标。