甲基化沉默的候选抑癌基因ZNF154调控EMT抑制鼻咽癌转移的机制

Distant metastasis is the main cause of treatment failure for nasopharyngeal carcinoma (NPC), DNA methylation alteration play an important role in carcinogenesis and development of NPC. In our preliminary studies, we found that methylation level showed significant differences in promoter region of zinc finger protein ZNF154 between the metastatic and non-metastatic NPC used the methylation microarray, and the methylation level are positive correlation with the metastasis in NPC. Further studies demonstrated that low expression of ZNF154 were always found in undifferentiated cell lines , and overexpression of ZNF154 could inhibit the invasion in tumor cell and significantly increase expression of E-cadherin proteins, the key molecular in process of epithelial-mesenchymal transition (EMT). While，EMT is the key step to tumor metastasis. Thus, we propose the scientific hypothesis that: ZNF154 with methylated silence might inhibit NPC invasion and metastasis through regulation of EMT. This project will: ① make a clear relationship between the methylation level and expression level of ZNF154 gene and metastasis in NPC, and verify its clinical application value; ② clarify biological function of ZNF154 and molecular mechanisms that ZNF154 inhibits metastasis by regulation of EMT in NPC. Ultimately, we will not only clear ZNF154 biological function, and identify a new biological marker for early diagnosis and prognosis of NPC, can also provide new targets for the prevention and treatment in NPC.

远处转移是鼻咽癌治疗失败的主要原因，DNA甲基化改变在鼻咽癌发生及转移过程中扮演重要角色。前期研究中，我们利用甲基化芯片检测发现转移与非转移的鼻咽癌组织中锌指蛋白ZNF154启动子区甲基化存在显著差异，且甲基化程度与鼻咽癌的转移呈正相关。ZNF154在低分化细胞株中表达显著下调，过表达ZNF154可抑制细胞侵袭，显著上调上皮间质转化（EMT）的关键抑制因子E-钙粘连蛋白。而EMT是肿瘤转移的关键步骤。据此提出：“甲基化修饰的ZNF154调节EMT抑制鼻咽癌的侵袭转移”的假说。为阐明该假说，本项目拟开展：①进一步明确ZNF154基因的甲基化程度及表达水平与鼻咽癌转移的关系，验证其临床应用价值；②阐明ZNF154的生物学功能及调节EMT抑制鼻咽癌转移的分子机制。项目的完成不但可以揭示ZNF154的生物学功能，还可为鼻咽癌早期诊断或预后提供新的生物学标志，为鼻咽癌的防治提供新靶标。

背景：鼻咽癌（NPC）高发于东南亚和中国南部，其发病分子机制仍有待进一步阐明。DNA甲基化与包括鼻咽癌在内的肿瘤的发生和发展相关。通过全基因组DNA甲基化测序方法，我们发现了ZNF154，但是它的甲基化状态和在鼻咽癌中的作用还有待阐明。.方法：用甲基化法检测鼻咽癌组织中ZNF154的甲基化状态特异性PCR（MSP）和定量序列分析。通过划痕实验和transwell实验观察其对鼻咽癌细胞迁移和侵袭的影响。通过观察细胞体内成瘤、转移情况变化，阐明ZNF154在体内对鼻咽癌转移的影响。通过Western blotting分析ZNF154过度表达后相关蛋白的表达变化。采用Kaplan-Meier分析确定ZNF154甲基化与鼻咽癌预后的关系。.结果：与正常鼻咽组织和细胞相比，ZNF154在鼻咽癌组织和细胞系中，由于启动子甲基化导致其表达水平下降。5-氮杂-2-脱氧胞苷（5-氮杂）脱甲基处理可恢复鼻咽癌细胞株ZNF154的表达。ZNF154基因异位高表达鼻咽癌细胞体外抑制细胞迁移和侵袭及体内肿瘤转移试验肺结节形成。机制研究表明ZNF154抑制Wnt/β-catenin信号通路的激活并抑制鼻咽癌上皮间质转化。此外，Kaplan-Meier分析显示ZNF154的高甲基化启动子与明显较差的无病生存率和无远处转移生存率显著相关。.结论：综上所述，这些结果表明ZNF154可作为鼻咽癌的潜在肿瘤抑制因子。