二亚硝基哌嗪（DNP）活化HSP70-2参与鼻咽癌转移的分子机制研究

Our previous works found that N,N'- dinitrosopiperazine (DNP) mediates nasopharyngeal carcinoma (NPC) through HSP70-2, following an increased HSP70-2APTase activity. Molecular dynamics simulations data showed that DNP binds to HSP70-2-J-domain. Based on these, we speculate that DNP may increase HSP70-2APTase activity through binding to HSP70-2-J-domain, and promotes NPC metastasis. In this project, we want to ①analyze the relationship of DNP concentration, HSP70-2APTase activity and NPC metastasis, and confirm whether DNP-mediated HSP70-2APTase activity involves NPC metastasis; ②construct HSP70-2-J-domain plasmids, transfect them to Hsp70-2-/- cells, analyze the interaction of 3H-DNP and J-domain, HSP70-2APTase activity and cell metastasis; ③construct HSP70-2-J-domain mutant dominant plasmids, transfect the plasmid to NPC cells, analyze DNP-mediated NPC metastasis when DNP dose not interact with J-domain; ④transfect HSP70-2 and its mutant dominant plasmids into Hsp70-2-/- cell, and the stable cell lines containing J-domain, HSP70 mutant dominant were obtained, these stable cell line were injected into nude mice, and DNP-mediated NPC metastasis though binding to J-domain-HSP70-2 were confirmed using animal experiments. The mechanism of DNP-mediated NPC metastasis though activating HSP70-2-APTase will be clarified, these will provide a novel clue for NPC prevention and therapy.

前期研究发现DNP通过HSP70-2促进鼻咽癌转移，HSP70-2APTase活性显著增高，分子模拟预测DNP与HSP70-2-J-domain结合。由此推测DNP与HSP70-2-J-domain结合，增强HSP70-2APTase活性参与鼻咽癌转移。本项目拟研究：①分析DNP浓度、HSP70-2APTase活化与鼻咽癌转移关系；②构建HSP70-2-J-domain质粒，转染Hsp70-2-/-细胞，分析3H-DNP与J-domain结合、HSP70-2APTase活化和细胞转移；③构建HSP70-2-J-domain突变体，分析DNP不与J-domain结合对HSP70-2APTase活性和细胞转移的影响。④将HSP70-2及突变体导入Hsp70-2-/-细胞，接种至裸鼠，动物实验分析细胞转移。阐明DNP通过活化HSP70-2APTase参与鼻咽癌转移的机理，为鼻咽癌防治提供科学依据

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。项目前期研究发现，鼻咽癌致癌因子二亚硝基哌嗪（DNP）参与了鼻咽癌的转移，但其作用的分子机理未阐明。本项目按《项目计划书》的研究计划，采用分子模拟技术、基因表达和基因干扰技术、免疫组织化学、蛋白印迹技术和动物实验，完成了项目的研究内容，达到了既定的研究目标，获得以下的研究成果：①检测鼻咽癌细胞和鼻咽癌组织HSP70-2APTase水解活性，发现HSP70-2-APTase水解活性与鼻咽癌转移的关系；分析DNP对鼻咽癌细胞HSP70-2-APTase水解活性和细胞侵袭转移能力的影响，发现DNP上调HSP70-2-APTase水解活性；②采用分子动态模拟技术分析DNP与HSP70-2结合，发现DNP与HSP70-2结构中J-domain结合。采用PCR技术构建含HSP70-2不同片段表达载体，采用基因转染技术将HSP70-2表达载体转染至鼻咽上皮细胞，采用免疫共沉淀+APase活性检测技术检测HSP70-2APTase水解活性。③成果构建了pcDNA3.1-Hsp70-2-j、pcDNA3.1-Hsp70-2-jM、pGEX-5x-Hsp70-2-j、pGEX-5x-Hsp70-2-Jm。④建立了免疫共沉淀+APase活性检测技术检测HSP70-2-APTase活性，核素标记DNP获得3H-DNP。 结果发现，HSP70-2-APTase水解活性与鼻咽癌转移的关系；⑤放射自显影发现DNP与HSP70-2结构中J-domain结合。⑥动物实验表明DNP通过HSP70-2-J-domain诱导鼻咽癌细胞体内转移。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6篇，会议论文2篇，培养研究生3名，其中博士1名，硕士2名。