二亚硝基哌嗪（DNP）介导Clusterin表达参与鼻咽癌转移的分子机制研究

Our previous works indicated that DNP mediates metastasis of nasopharyngeal carcinoma (NPC) 6-10B cells, following high expressions of clusterin(CLU), cathepsin, AGR2, and cytoskeletal protein.And, motility and invasion of NPC cells transfected with CLU siRNA could not effectively induce by DNP in previous trials. CLU directly or indirectly regulated MMP-9 and VEGF expression in tumor invasion and metastasis. So,we speculate that DNP induces CLU expression through MMP-9 and VEGF expression, increases the motility and invasion of cells, and promotes NPC tumor metastasis. In this subject, we intend to conduct the following research:① analyzing that DNP mediated metastasis of nasopharyngeal carcinoma 6-10B cells through CLU;② analyses the interaction of DNP and the promoter region of AP1 using Molecular dynamics Simulations;③ building promoter AP1 carrier of CLU, analyzing that binding between DNP and AP1 effect on the expression of CLU;④analyzing that the motility and invasion of NPC cells,and MMP-9, and VEGF expression with CLU siRNA;⑤ building 6-10B-siCLU,and injecting into the tail veins of nude mice. Simultaneously, DNP were abdominally injected into these mice, Will be evaluating DNP on NPC metastasis in nude Mice. As a result, we expect our study will improve the study of machanism of DNP mediated metastasis of nasopharyngeal carcinoma, and provide new targets for comprehensive therapy.

前期研究发现DNP促进鼻咽癌转移，诱导Clusterin(CLU)高表达， CLU表达沉默显著降低DNP诱导鼻咽癌转移。CLU诱导MMP-9、VEGF表达参与肿瘤转移。由此推断DNP可能上调CLU介导MMP-9、VEGF表达参与鼻咽癌转移。本项目拟进行以下研究：①分析DNP诱导鼻咽癌细胞CLU表达与细胞侵袭转移；②分子模拟预测DNP与CLU启动区AP1结合（已完成）；③构建CLU启动子AP1载体，分析DNP与AP1结合依据其对CLU表达的影响；④分析siRNA阻断CLU对DNP诱导细胞侵袭转移和MMP-9、VEGF表达的影响；⑤构建siCLU细胞，接种至裸小鼠并DNP处理，动物实验验证DNP通过CLU促进鼻咽癌转移。本项目完成将获得DNP通过 CLU/MMP-9-VEGF/metastasis参与鼻咽癌转移的分子机制，为鼻咽癌转移防治提供科学的理论依据。

鼻咽癌具有高转移的临床特征，二亚硝基哌嗪（N，N’- Dinitrosopiperazine，DNP）作为一种化学致癌因子参与了鼻咽癌的转移，但其确切机制尚未完全阐明。我们研究发现，DNP通过上调丛生蛋白（clusterin，CLU）的表达促进鼻咽癌转移。进一步研究发现，DNP通过上调CLU的表达增加MMP-9和VEGF表达，同时，可诱导MMP-9和VEGF结合，进而诱导鼻咽癌细胞运动和侵袭。病理研究也发现，CLU，MMP-9和VEGF表达与肿瘤的（TNM）分期呈正相关。这些结果表明，DNP通过上调CLU介导MMP-9和VEGF的表达促进鼻咽癌肿瘤转移，CLU的表达是DNP诱导鼻咽癌转移的一个重要因素。