EB病毒癌蛋白LMP1抑制miR-449b-3p促进鼻咽癌转移的机制研究

We have identified miR-449b-3p as a metastasis-related non-coding RNA that is frequently downregulated in the nasopharyngeal carcinoma (NPC) tissues using miRNA microarray and TCGA database analyses followed by validation with quantitative PCR. Our preliminary data revealed that downregulation of miR-449b-3p is inversely correlated with EBV-DNA copy number and 2008 TNM stage and is associated with poor prognosis. Overexpression of miR-449b-3p significantly suppressed NPC cell proliferation, invasion and migration. Interestingly, we found that miR-449b-3p expression was inhibited by LMP1, an Epstein-Barr virus-encoded oncoprotein, and that ADAM17, a protein strongly correlated with tumor cell invasion, migration and poor prognosis, is a direct target of miR-449b-3p in NPC cells. Together, our results reveal a novel hypothesis that LMP1 induces ADAM17 expression and promotes NPC cell invasion and migration via suppressing miR-449b-3p. This proposal is designed to further investigate the LMP1/miR-449b-3p/ADAM17 interaction network and its implication in NPC. Multidisciplinary approaches including gain- and loss-of-function analysis in cell and in vivo, rescue experiments, luciferase reporter assays, RIP-chip assays and promoter methylation detection will be performed. Finally, we will comprehensively analyze the clinical correlation between LMP1/miR-449b-3p/ADAM17 axis and NPC progression. Thus, our novel finding would not only provide a molecular insight into how EBV oncoprotein LMP1 interacts with host-miRNA, but also contribute to the development of new biomarker and therapeutic target in NPC.

基于基因芯片及TCGA大数据分析筛选，我们发现miR-449b-3p差异低表达于转移性鼻咽癌，验证显示miR-449b-3p在鼻咽癌组织中低表达，与EB病毒DNA拷贝数、肿瘤分期负相关，与患者预后正相关；过表达miR-449b-3p可抑制细胞增殖、侵袭迁移。预实验发现EB病毒癌蛋白LMP1负调控miR-449b-3p表达，而与肿瘤侵袭转移及不良预后密切相关的ADAM17为miR-449b-3p的下游靶基因。故提出“LMP1通过抑制miR-449b-3p诱导ADAM17表达促进鼻咽癌侵袭转移”的科学假说。本项目拟在体内外水平采用过表达/沉默策略，通过拯救实验、报告基因、RIP-Chip、启动子甲基化等实验，结合临床验证，阐明miR-449b-3p受LMP1调控并通过下游ADAM17参与鼻咽癌侵袭转移的分子机制。本假说的阐明，可为EB病毒与宿主miRNA的相互调控及鼻咽癌防治提供新靶标。

研究背景. EBV与鼻咽癌发生发展密切相关，从EBV与宿主的非编码基因表达失衡角度，继续发现鼻咽癌侵袭转移的新靶标并深入探讨miRNA调控机制，对阐明NPC的发病机制至关重要。.研究内容. 利用NPC组织和细胞检测miR-449b-3p的表达，使用统计学方法分析miR-449b-3p的表达与NPC患者临床病理特征的相关性及其与预后的关系。使用CCK8,克隆平板形成，细胞划痕和Transwell侵袭与迁移等实验分析miR-449b-3p对细胞功能变化的影响。使用PCR检测LMP1对miR-449b-3p的影响，利用荧光素酶，CHIP实验分析LMP1对miR-449b-3p的表达调控机制。使用在线数据库分析miR-449b-3p的可能靶基因，利用荧光素酶实验验证两者相关性。在体内动物水平分析miR-449b-3p对NPC转移的作用。.研究结果. 组织内miR-449b-3p在NPC组织内低表达，其表达水平与NPC患者EBV-DNA拷贝数及临床分期呈负相关；且miR-449b-3p表达越高，预后越好。体外细胞功能实验显示miR-449b-3p有效抑制NPC的侵袭与迁移能力，但是对细胞的增殖凋亡作用不明显，体内动物实验证明miR-449b-3p可明显抑制NPC细胞的转移。EBV编码癌蛋白LMP1可在转录水平激活NF-κB信号通路抑制miR-449b-3p的表达；下游机制分析发现miR-449b-3p可靶向抑制ADAM17的表达。进一步的挽救实验结果证明 miR-449b-3p可以通过靶向结合ADAM17，逆转细胞的EMT进程，抑制NPC细胞的侵袭与迁移能力。.研究意义.本研究的成功实施，将为鼻咽癌侵袭转移进展的miRNA调控模式提供新的理论依据，进而为鼻咽癌临床诊疗提供新的靶点。