WIPI-1启动子低甲基化在鼻咽癌转移中的作用及机制研究

Distant metastasis is the main cause of treatment failure and death in nasopharyngeal carcinoma (NPC). Thus, clarification of the molecular mechanisms of distant metastasis, and identification of effective molecular markers associated with prognosis prediction and targeted therapy are crucial to further improve the treatment outcome of NPC. To date, aberrant DNA methylation has been found to play an important role in the development and progression of NPC. Our previous study found that: ① DNA methylation status was significantly different between metastatic and non-metastatic NPC tissues. WIPI-1 methylation level was significantly lower in metastatic NPC tissues; ② patients with WIPI-1 hypomethylation had significantly higher risk for distant metastasis; ③ hypomethylation of WIPI-1 was associated with increased mRNA expression, and siWIPI-1 suppressed the migration and invasion of NPC cells and inhibited the ability to resist anoikis and epithelial-to-mesenchymal transition (EMT) in vitro; however, the mechanism remains unknown. In this study, we will detect the WIPI-1 methylation status in 400 NPC patients and examine its prognostic value for distant metastasis. Furthermore, we will study the functions and mechanisms of WIPI-1 in vitro and in vivo. Through this research, it may provide a potential novel target for molecular classification and individual therapy in NPC patients.

远处转移已成为制约鼻咽癌生存率提高的重要因素，阐明鼻咽癌远处转移的分子机制，寻找有效的预后指标和治疗靶点，是进一步提高疗效所需要解决的关键科学问题。鼻咽癌的发生发展与DNA启动子区域异常甲基化密切相关，我们的前期研究显示：①有远处转移与无远处转移的鼻咽癌组织的甲基化状态存在显著差异，其中，WIPI-1的甲基化水平在有远处转移的鼻咽癌组织中显著降低；②WIPI-1低甲基化患者的远处转移风险明显升高；③WIPI-1低甲基化使其在鼻咽癌细胞中表达增加，而瞬转敲减WIPI-1后可显著抑制鼻咽癌细胞的迁移、侵袭能力，并降低其抗失巢凋亡能力和上皮间质转化水平，但具体作用机制不详。本项目拟在此基础上，检测400例鼻咽癌组织中WIPI-1启动子甲基化状态，进一步明确其在鼻咽癌远处转移中的预后价值；同时通过体内外模型阐明WIPI-1在鼻咽癌转移中的作用及机制，为鼻咽癌分子分型和个体化治疗提供新的潜在靶点。

背景：鼻咽癌在头颈部肿瘤中的转移率最高，高发于华南地区，又称“广东溜”。放射治疗是目前鼻咽癌治疗的主要手段，而远处转移是鼻咽癌治疗失败和死亡的主要原因。因此，需要新的生物标志物来设计新的治疗策略以改善预后。WIPI-1在多种肿瘤中异常表达。然而，WIPI-1在鼻咽癌中的表达水平和潜在功能及分子机制尚不清楚。.方法：采用qRT-PCR和westernblot方法检测基因表达。随后采用Transwell法、集落形成法和CCK-8法检测WIPI-1对鼻咽癌细胞的体外迁移和增殖能力的影响。采用裸鼠尾静脉-肺转移模型、足底腘-淋巴结转移模型移植瘤模型和裸鼠皮下移植瘤模型，评价WIPI-1在体内对鼻咽癌细胞转移和增殖能力的影响。采用免疫共沉淀结合质谱，探究WIPI-1下游相互作用蛋白，并用免疫共沉淀、免疫荧光染色进行下游靶点验证。利用RNA测序，挖掘WIPI-1下游信号分子，探究鼻咽癌中WIPI-1可能的调控机制。.结果：在鼻咽癌细胞和组织中，WIPI-1的表达明显降低。此外，WIPI-1低表达与鼻咽癌患者预后不良密切相关。体外功能实验表明，过表达WIPI-1抑制了鼻咽癌细胞的迁移、集落形成和增殖能力；相反的，敲低WIPI-1促进鼻咽癌细胞的迁移、集落形成和增殖能力；与体外研究相一致，通过体内窝淋巴结转移、肺转移和皮下移植模型，发现WIPI-1显著抑制鼻咽癌细胞的生长、体内侵袭和转移。机制研究发现，WIPI-1与TRIM21存在直接相互作用；并且其通过与TRIM 21相互作用，增强了鼻咽癌细胞中由饥饿诱导的自噬反应。此外，通过RNA-seq分析，WIPI-1敲除组的MYC基因表达明显显著上调。QPCR验证发现过表达WIPI-1抑制MYC基因表达和MYC报告基因活性，而敲低WIPI-1显著促进MYC基因表达和MYC报告基因活性。.结论：WIPI-1在鼻咽癌中扮演着抑癌基因的角色，通过与TRIM21相互作用，促进自噬反应；同时抑制MYC基因活性及表达，抑制肿瘤的生长和转移。鉴于WIPI-1低表达导致鼻咽癌预后差，靶向WIPI-1可能是鼻咽癌治疗的一种新途径。