二亚硝基哌嗪上调AGR2促进鼻咽癌细胞转移

Dinitrosopiperazine-mediated up-regulation of AGR2 expression promotes metastasis of nasopharyngeal cancer cells

目的：探讨二亚硝基哌嗪（DNP）通过调控AGR2表达参与鼻咽癌转移的分子机制。方法：以具有低转移潜能的鼻咽癌细胞6-10B作为研究材科,应用四甲基噻唑蓝（MTT）法检测DNP对6-10B细胞的非毒性浓度;分别采用间接免疫荧光法、Western blot和实时荧光定量PCR法检测DNP对6-10B细胞中AGR2蛋白及m RNA表达的影响;采用针对AGR2基因的特异性小干扰RNA（si RNA）沉默6-10B细胞AGR2的表达,并用Transwell小室法检测其对细胞侵袭和迁移能力的影响。结果：MTT法检测DNP对6-10B细胞的非毒性浓度为0-10.0μmol/L;8.0μmol/L DNP处理6-10B细胞后,间接免疫荧光法检测发现AGR2蛋白表达明显上调,且以在细胞质中表达为主;不同浓度的DNP处理6-10B细胞24 h或用8.0μmol/L的DNP处理6-10B细胞不同时间后,Western blot结果发现AGR2蛋白的表达水平增加且呈浓度和时间依赖性（P〈0.05）;siRNA-AGR2沉默6-10B细胞屮AGR2基因的表达后,DNP诱导6-10B细胞的侵袭及迁移能力较干扰前明显降低（P〈0.05）。结论：DNP可能通过在转录水平上调AGR2的表达,影响鼻咽癌细胞的侵袭和转移能力。

OBJECTIVE： To investigate theinduction of AGR2 expression by dinitrosopiperazine（DNP） in nasopharyngeal cancer cell（NPC） 6-10 B and to explore involvement of DNP in NPC metastasis. METHODS：Noncytotoxic concentrations of DNP on 6-10 B cells were determined using MTT. Expression of AGR2 protein and m RNA in 6-10 B cells which were treated with DNP were detected by indirect immunofluorescence,Western blotting and real-time RT-PCR,respectively. In addition,silencing the expression of AGR2 by siRNA-AGR2 was investigated. The capabilities of invasion and migration of 6-10 B cells were investigated using Transwell assay. RESULTS：The range of non-cytotoxic concentrations（NCC） of DNP was 0-10.0 μmol/L. The results of indirect immunofluorescence showed AGR2 mainly expressed in cytoplasm after DNP treatment. These and 5-8F cells had stronger fluorescence intensity than untreated 6-10 B cells. The DNP-induced AGR2 expression showed dose- and time-dependent effects. Real-time RT PCR analyses showed that AGR2 m RNA expression rate was 1.01±0.08 in the untreated 6-10 B cells and 3.96±0.15 in the cells treated with 8.0 μmol/L DNP. The difference was significant. In addition,the relative fold gene expression of AGR2 in 5-8F cells was higher than that in non-treated 6-10 B cells（P〈0.05）. The transwell migration and invasion data showed that si RNA-AGR2 transfection blocked AGR2 expression in 6-10 B cells. In addition,both DNP-induced invasion and motility were dramatically decreased when AGR2 expression was blocked（P〈0.05）. However,DNP could effectively induce cell invasion（P〈0.05） and motility（P〈0.05） when AGR2 was not blocked. CONCLUSION：DNP could up-regulate AGR2 expression in 6-10 B cells through transcriptional regulation mechanism and then mediate the metastasis of nasopharyngeal cancer cells.