EBERs对鼻咽癌转移的作用及其机制研究

EBERs promote metastasis of nasopharyngeal carcinoma via epithelial-mesenchymal transition

摘要:

目的  探讨EBERs对鼻咽癌转移的作用及其机制。方法  在鼻咽癌细胞中转染EBERs，荧光定量PCR（RT-PCR）检测细胞中转移相关指标（如MMP）的相对表达量，同时 Transwell检测EBERs对鼻咽癌细胞迁移能力的影响。构建敲除EBERs的BAC-EBV（p2089）质粒，转染鼻咽癌细胞，检测细胞的转移能力。转染EBERs后Western blot检测上皮间质转化（EMT）相关指标，免疫荧光检测ZO-1的表达。结果  鼻咽癌细胞转染EBERs质粒或p2089质粒后，RT-PCR检测发现转移相关指标表达升高，Transwell检测结果显示细胞迁移能力增强，而在干扰EBERs或转染敲除EBERs的p2089质粒后，转移能力减弱。转染EBERs后E-cadherin表达降低，Vimentin升高，同时免疫荧光检测发现ZO-1表达降低。结论  EBERs可能通过促进EMT高表达而促进鼻咽癌的转移。

Abstract:

Objective To investigate whether EBV encoded RNAs (EBERs) could promote metastasis of nasopharyngeal carcinoma (NPC) and the possible involvement of epithelial-mesenchymal transition (EMT) in this process. Methods EBERs expression plasmid was transfected into EBV negative NPC cell lines with lipofectamine 2000. 48 h after transfection, matrigel migration assay and cell counting were conducted to explore the impact of EBERs on NPC migration and proliferation, respectively. In addition, siRNA targeting EBERs was applied to EBV positive C666-1 cells, after that the expression changes of metastasis related genes were detected. To consolidate the observation, EBERs-deleted EBV expression plasmid was contructed based on bacterial artificial chromosome (BAC) system. Then expression changes of metastasis related genes were compared between the cells harboring EBV with or without EBERs. Futhermore, the involvement of EMT associated genes in EBERs-mediated metastasis was explored via Western blot, and immunofluorescence was used to detect ZO-1 expression. Results Transient transfetion of EBERs significantly improved metastasis of NPC cells and expression of metastasis related genes. However, EBERs had no impact on NPC cell proliferation. In support of the observation, siRNA targeting EBERs or EBERs deletion significantly decreased expression of metastasis related genes. Furthermore, EBERs expression decreased ZO-1 and E-cadherin expressions while increased Vimentin expression. Conclusions EBERs could promote metastasis of NPC cells, and EMT probably contributes to this process.