锌指蛋白ZNF146表达下调在鼻咽癌增殖中的作用及分子机制研究

Previously, we analyzed a large samples of nasopharyngeal carcinoma (NPC) gene expression microarray dataset and found that ZNF146 is the hub node of the transcriptional regulatory networks in NPC. Immunohistochemistry analysis showed that ZNF146 was strong expressed in all of the 61 cases of normal nasopharyngeal (NP) tissues tested, but only highly expressed in 31 (17.5%) out of the 177 cases of NPC tissues tested. The expression of ZNF146 was related with the TNM stages of NPC. The knockdown of ZNF146 in NPC cell lines was found to promote the proliferation and stemness of NPC cells. Taken together, it seems that ZNF146 is important for initiation and progression of NPC. To validate this hypothesis, we plan to: 1) investigate the roles of ZNF146 on the proliferation and stemness of NPC, and the resistance of radiotherapy for NPC with the in-vitro and in-vivo methods; 2) reveal the regulatory networks for ZNF146 involved in regulation of proliferation, stemness, and radiotherapy-resistance of NPC with high throughput methods; 3) screen and validate the targeted genes of ZNF146 with the ChIP-seq and RNA-seq methods; 4) predict and validate the transcription factor that regulate the expression of ZNF146 in NPC; 5) confirm whether the down-regulation of ZNF146 expression in NPC was due to promoter hypermethylation; 6) reveal the relationship between the involved pathways of ZNF146 and the clinical parameters of NPC patents. We hope that this project can provide theoretical foundation for developing new diagnosis and treatment strategies for NPC.

申请人前期分析大样本鼻咽癌（NPC）基因芯片数据发现，ZNF146是NPC转录调控网络的核心节点；免疫组化揭示ZNF146在61例鼻咽非癌组织中全部强阳性表达，而在177例NPC标本中仅有17.5%标本高表达且与TNM分期相关；沉默ZNF146促进NPC细胞增殖和增强干性；以上信息提示，ZNF146与NPC发生发展可能有着密切关系。据此，本研究拟开展以下研究：1)ZNF146对NPC细胞增殖、干性和放疗敏感性的影响；2)高通量方法揭示ZNF146影响NPC细胞生物学特性的调控网络；3)ChIP-seq和RNA-seq筛选ZNF146靶基因及功能验证；4)软件预测上游直接调控ZNF146的基因及功能验证；5)确证启动子甲基化与NPC中ZNF146表达下调的相关性；6)利用人NPC临床组织标本，揭示ZNF146及其上下游基因的表达与临床病理意义。本研究有望为发展新的NPC诊治策略提供理论依据。

鼻咽癌（nasopharyngeal carcinoma，NPC）是我国南方高发的恶性肿瘤，主要治疗手段是放疗和化疗。但是放化疗尤其是放疗存在着严重的头颈部并发症，晚期远处转移病人仍然缺乏有效的治疗手段。因此，阐明NPC进展的分子机制，为发展新的治疗手段和策略提供理论依据，具有重要意义。转录因子在调节基因表达过程中发挥关键作用，其异常是癌症发生发展的主要原因之一，因此也是癌症诊治靶标的研究热点。. 我们分析大样本NPC基因芯片数据发现，ZNF146是NPC转录调控网络的核心节点；免疫组化揭示ZNF146在61例鼻咽非癌组织中全部强阳性表达，而在177例NPC标本中仅有17.5%标本高表达且与TNM分期相关；体内外实验表明ZNF146抑制NPC细胞增殖和干性。结合CHIP-seq和RNA-seq分析发现ZNF146作为转录抑制因子，下调与NPC干性、增生和迁移相关基因。进一步地，ZNF146通过c-jun蛋白调控NPC干性、增生和迁移。. 我们通过甲基化测序分析发现启动子甲基化不是ZNF146在NPC中表达下调的原因。相反，结合生物信息学预测和实验验证发现E2F1是上游直接调控ZNF146的转录因子。在NPC组织标本中，我们发现E2F1、ZNF146和c-jun表达显著相关。因此，E2F1-ZNF146-JUN轴在NPC的发生发展过程中发挥重要作用。进一步地，在NPC PDX模型中，我们发现过表达ZNF146可以有效抑制肿瘤生长，因此ZNF146可以作为NPC的治疗靶点。.