二亚硝基哌嗪（DNP）通过CTSB调控EMT参与鼻咽癌转移的分子机制研究

Based on DNP (N,N-Dinitrosoperazine,DNP) promoting nasopharyngeal carcinoma (NPC) metastasis, upregulating expression of cathepsin B and vimentin biomarker for epithelial-mesenchymal transition（EMT）, and molecular simulation predicting the binding of DNP and CTSB-papain fold in the previous works. We can drawn from a hypothesis: DNP mediates the high expression of CTSB through binding CTSB-papain, high CTSB upregulating EMT participates in NPC metastasis. In this project, the molecular mechanisms of DNP regulation of EMT through cathepsin B involved in nasopharyngeal carcinoma metastasis are to be investgated using molecular simulation, siRNA blocking and metastasis assay in nude mice.We are to perform the follow: ①Investigating the relationship of CTSB expression, EMT and NPC metastasis in clinical; ②The interaction of DNP and CTSB is analyzed using molecular simulation, it found DNP binding to the papain domain of CTSB, and increasing CTSB expression. These findings are proved using the experiments; ③analyzing the regulation of invasion,metastasis and EMT mediated by DNP through CTSB. ④using siRNA to block cathepsin B, and then testing DNP regulation to invasion, metastasis and EMT under cathepsin B blocked;⑤ DNP regulation of EMT through cathepsin B are proved by metastasis assay in nude mice. In this present,we will clarify that DNP mediates NPC metastasis through cathepsin B regulation of epithelial-mesenchymal transition.

基于前期研究发现DNP促进鼻咽癌（NPC）转移并上调cathepsin（CTSB）和EMT标志物 Vimentin表达，分子模拟发现DNP与CTSB-papain结合。由此推断：DNP通过与CTSB结合增强CTSB表达，高CTSB调控EMT参与NPC转移。本项目拟采用分子模拟、siRNA干扰、动物实验等探讨DNP通过CTSB调控EMT参与NPC转移分子机制研究，拟开展1）检测NPC组织和转移组织CTSB及EMT标志物表达，明确其与NPC转移的关系；2）分子模拟技术分析DNP与CTSB结合，发现DNP与CTSB-papain fold结合。实验验证DNP与CTSB结合，增强CTSB表达；3）分析CTSB对细胞EMT、侵袭与转移的调控; 4) siRNA阻断CTSB，分析DNP对细胞EMT和侵袭转移调控；5)动物实验确证DNP上调CTSB介导EMT促进NPC转移。阐明鼻咽癌高转移的分子机制。

本课题在细胞和组织鼻咽癌组织和细胞中的CTSB和 CTSD水平显著高于正常鼻咽细胞和组织，且其升高水平和化学致癌物DNP呈正相关，进一步研究发现DNP可促进CTSB的表达且与鼻咽癌的恶性程度呈正相关，采用ELISA方法检测健康人群、鼻咽癌患者血清中CTSB和 CTSD浓度，发现鼻咽癌转移者血清中CTSB和 CTSD浓度高于健康人群，CTSB和 CTSD水平与鼻咽癌转移密切相关，但与鼻咽癌患者的预后没有显著的相关性。基于此研究基础，将研究方向作了微调，应用小 RNA 测序技术筛选鼻咽癌细胞 5-F、 DNP -6-10B和 6-10B三组细胞间 差异表达 miRNAs，对其中 20 个高表达的 miRNAs进行了定量 RT -PCR的验证， 结果与测序的一致，且发现 miR -141 -3p 的差异最明显，且在鼻咽活检组织中证实 miR -141 -3p 在鼻咽癌组织中的表达明显高于正常鼻咽组织，荧光素酶双报告实验证实PRKAR1A为miR -141 -3p靶基因，进一步通过高表达和低表达miR -141 -3p证实，高表达的 miR -141 -3p抑制癌基因 PRKAR1A表达，促进鼻咽癌的增殖和侵 表达，促进鼻咽癌的增殖和侵 袭转移 ，提示 miR -141 -3p 参与了鼻咽癌的侵袭转移。