鼻咽癌（NPC）高转移严重影响NPC有效治疗，前期研究发现DNP诱导NPC转移伴AGR2高表达，分子模拟显示DNP与Agr2启动子结合，阻断Agr2基因，Cathepsin B、D表达下降，细胞转移降低。我们推断：DNP上调AGR2介导Cathepsin B、D表达，促进NPC转移。本项目拟进行: ①分析DNP诱导NPC细胞转移及AGR2、Cathepsin B、D表达；②分析沉默Agr2对DNP诱导Cathepsin B、D表达和细胞转移的影响及沉默Cathepsin b,d基因是否抑制DNP诱导细胞转移；③DNP对Agr2表达调控启动子分析；④动物实验分析Agr2、Cathepsin b或d 沉默DNP介导的细胞转移。⑤分析NPC患者DNP、AGR2与肿瘤转移的关系。阐明DNP通过 AGR2/Cathepsin B、D参与NPC转移的分子机制，为NPC转移分子机理研究提供科学理论依据

The metastasis of nasopharyngeal carcinoma (NPC) impact NPC therapy. Our previous works found that Dinitrosoperazine (DNP) induces NPC metastasis with a high expression of Anterior gradient-2 (AGR2)，molecule simulation showed that DNP interacts with the promoter of Agr2 gene, while Cathepsin B,D expression and cell metastasis significantly decrease while Agr2 blocked. We speculate that DNP may up-regulate AGR2, mediate Cathepsin B、D expression, and promote NPC metastasis. In this study, ① AGR2,Cathepsin B and D expressions are detected in NPC cells with DNP treatment and its metastasis is also measured. ② Cathepsin B,D expressions are detected in the Agr2 -blocked NPC cells and its metastasis is measured after DNP treatment. The cell metastasis is also detected when Cathepsin b or d gene blocked. ③ Agr2 gene promoter activity is analyzed followed DNP treatment. ④ When Agr2、Cathepsin b or d gene is blocked, DNP-mediated cell metastases are tested using nude mice. ⑤The relationships of DNP concentration, AGR2 level and NPC metastasis are analyzed in NPC patients. We will prove DNP-AGR2-Cathepsin B、D-metastasis signal pathway. This will provides a novel clue for NPC metastasis research.

鼻咽癌（NPC）具有高转移、高复发的临床特征，给鼻咽癌的有效治疗带来极大的难题。本项目采用分子模拟、基因表达和基因干扰等分子生物技术研究DNP参与鼻咽癌转移的机制，获得以下的研究成果：1）AGR2表达在鼻咽、原发部位鼻咽癌组织和淋巴结转移组织逐步增高，AGR2表达与CTSB、CTSD呈正相关。鼻咽癌转移患者尿DNP高于无转移者和健康人群，且DNP水平与转移密切相关。2)采用低转移鼻咽癌细胞株6-10B细胞作细胞模型, 分析DNP对细胞AGR2、CTSB和CTSD表达的影响，发现DNP促进AGR2、CTSB和CTSD表达，且呈浓度和作用时间依赖性。3）用Boyden-chamber assay分析细胞侵袭移动能力，Western-blotting 检测AGR2、Cathepsin B、D表达。结果发现DNP促进6-10B 细胞AGR2表达，促进侵袭移动。4）分子模拟推算DNP与调控元件XRE可能结合的化学键能，结果发现DNP与XRE1、2、3结合。5）根据分子模拟结果, 构建含不同XREs的AGR2基因启动子质粒。用QuikChange Ⅱsite-directed mutagenesis构建pcDNA3.1-AGR2 promoter突变体。核素自显影，发现DNP与含XRE的质粒DNA结合。6）荧光酶报导基因活性分析XRE对DNP诱导AGR2基因转录活性的影响。结果发现DNP通过XRE诱导AGR2基因表达。7）Western-blotting 分析CTSB、CTS D、MMP9、VEGF表达； Real-time PCR分析CTS B、CTSD基因表达；Flourimetric Innozenmy方法检测CTSB、CTSD酶活性。结果发现， DNP诱导鼻咽癌细胞CTSB、CTSD表达，其表达与AGR2的相关性。8）动物实验发现CTSB、CTSD在DNP诱导鼻咽癌细胞侵袭移动中起促进作用。这为进一步研究鼻咽癌转移的分子机制提供了新的思路。在本课题资助下，发表学术论文8篇，会议论文6篇，培养研究生8名，其中博士4名，硕士4名。