β-Trcp介导FOXK1泛素化修饰在鼻咽癌放疗抵抗中的机制研究

FOXK1 has been shown to be associated with outcomes of a variety of tumors, but its role in nasopharyngeal carcinoma remains unclear. β-Trcp is a ubiquitin protein, and preliminary experiment showed that FOXK1 interacts with β-Trcp1. Through the gene enrichment in the TCGA database, we found that FOXK1 was associated with ubiquitination significantly in nasopharyngeal carcinoma, and FOXK1 may affect the function of the nasopharyngeal carcinoma through JAK/STAT pathway. In addition, the expression of FOXK1 in nasopharyngeal carcinoma cells was lower than that in nasopharyngeal normal epithelial cell. The protein expression of FOXK1 increased significantly after the silencing of β-Trcp. Moreover, nasopharyngeal carcinoma cell growth was accelerated and radioresistance was increased after the silencing of β-Trcp. Accordingly, it was suggested that FOXK1 may be modified by β-Trcp ubiquitination to affect the development and radiotherapy sensitivity of nasopharyngeal carcinoma via the JAK/STAT pathway, which has not been reported at home and abroad. This study will investigate the ubiquitination of FOXK1 by β-Trcp, the function of FOXK1 in nasopharyngeal carcinoma and its relationship with radiotherapy resistance. Moreover, we will further clarify the clinical expression of FOXK1 in nasopharyngeal carcinoma. The study is important for understanding the development of nasopharyngeal carcinoma and the molecular mechanism of radiotherapy resistance, and may provide a new target for the treatment of nasopharyngeal carcinoma.

研究表明FOXK1与多种肿瘤预后相关，但在鼻咽癌中的功能尚不明确。β-Trcp是泛素化蛋白，预实验表明FOXK1与β-Trcp1有相互作用。通过TCGA数据库基因富集分析，发现FOXK1在鼻咽癌中与泛素化显著相关，并且FOXK1可能通过JAK/STAT通路影响鼻咽癌的功能。另外，FOXK1蛋白在鼻咽癌细胞的表达较鼻咽正常上皮细胞低，沉默β-Trcp后，FOXK1表达明显上升，鼻咽癌细胞生长加快、放疗抵抗增加，据此提出FOXK1可能被β-Trcp泛素化修饰，影响鼻咽癌的发生发展和经JAK/STAT通路影响放疗敏感性，目前此研究在国内外尚无报道。本课题将探讨β-Trcp对FOXK1的泛素化作用，以及FOXK1在鼻咽癌中的功能以及与放疗抵抗的关系，并进一步明确FOXK1在鼻咽癌中的临床表达水平。本课题的研究对了解鼻咽癌发生发展以及放疗抵抗的分子机制有重要作用，可能为鼻咽癌的治疗提供新的靶点。

目的：鼻咽癌是中国最常见的头颈部肿瘤。本实验旨在通过调节Janus kinas/Signal Translator and Actifator of Transfer 3(JAK/STAT3)通路，研究FOXK1干扰对鼻咽癌细胞增殖、凋亡、侵袭、上皮间质转化（EMT）和放射敏感性的影响。.方法：采用免疫组织化学方法检测临床鼻咽癌组织及鼻咽炎性组织中FOXK1的表达，并探讨其与肿瘤分期的关系。通过集落形成实验分别计算干扰FOXK1后和对照组的细胞集落形成率；流式细胞仪分别检测干扰FOXK1表达后和对照组细胞凋亡率和细胞周期分布；Transwell法检测细胞侵袭力；激光共聚焦显微镜下用免疫荧光法计数2Gy照射后30min、4h、24h各组细胞核内的 γ-H2AX焦点数。.结果：FOXK1在鼻咽癌患者癌组织中有明显表达，FOXK1的表达与性别显著相关，但与分期无明显相关。FOXK1干扰或FOXK1抑制剂Pefitinib可上调5-8F和CNE-2细胞的凋亡率，增加细胞的G2期比例，抑制肿瘤细胞的侵袭、迁移和EMT。同时，FOXK1干扰可下调细胞内p-JAK1、p-JAK2和p-STAT3和EMT相关蛋白如Snail、MMP-2、MMP-9的表达，增加Bax、Bcl-2凋亡蛋白的表达。单独干预FOXK1或Pefitinib可降低不同辐射剂量下的细胞集落形成率，并增加细胞核内γ-H2AX焦点的数目。此外，当FOXK1干扰和Pefitinib联合使用时，上述结果最佳。.结论：在鼻咽癌细胞，抑制FOXK1可通过JAK/STAT3信号通路调节EMT，增强细胞的放射敏感性，从而抑制肿瘤细胞的增殖和发展。