siRNAs干扰EBNA1下调EBV阳性鼻咽癌细胞内ALOX12的表达  

siRNAs Targeting EBNA1 Down-regulate ALOX12 Expression in EBV Positive NPC Cell Line

作　　者：刘文举[1] 王凤伟[1] 买世娟[1] 

机构地区：[1]华南肿瘤学国家重点实验室∥中山大学肿瘤防治中心实验研究部,广东广州510060

出　　处：《中山大学学报（医学科学版）》2010年第6期755-760,共6页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：国家重点基础研究计划(973)项目(2006CB910104)

摘　　要：【目的】利用RNA干扰技术抑制C666-1细胞中EB病毒核抗原1(EBNA1)的表达,探索氧化应激与抗氧化相关基因的表达变化。【方法】采用蛋白质印迹证实了能有效抑制EBNA1表达的干扰序列,使用甲基噻唑基四唑(MTT)方法检测C666-1细胞的存活率,利用实时定量PCR芯片技术探索了氧化应激与抗氧化相关基因的转录谱变化,并通过反转录聚合酶链反应(RT-PCR)和蛋白质印迹进行了验证。【结果】EBNA1-1414和EBNA1-1437干扰序列转染C666-1细胞48h后,分别可抑制59%和56%的EBNA1蛋白表达,细胞存活率分别降低了33%和24%。PCR芯片实验发现干扰EBNA1表达后48hC666-1细胞中花生四烯酸盐12-脂氧合酶(ALOX12)蛋白表达有下调,谷胱甘肽还原酶(GSR)和过氧化物酶3(PRDX3)在转录水平表达上调。【结论】EBNA1或许能够通过上调ALOX12的表达间接地发挥促鼻咽癌作用。【Objective】 To suppress Epstein-Barr virus nuclear antigen 1 （EBNA1） expression in C666-1 cells by using the RNA interference strategy,and investigate expression change of genes involved in oxidative stress and antioxidant defence response. 【Methods】 Small interfering RNAs targeting EBNA1 were verified by Western blot. The survival of C666-1 cells was measured by the methyl thiazolyl tetrazolium （MTT） assay. The transcriptional profiling of genes involved in oxidative stress and antioxidant defence response was analyzed by using real-time PCR-microarray method and the PCR-microarray result was verified by reverse transcription polymerase chain reaction （RT-PCR） and Western blot. 【Results】 EBNA1 protein expression was suppressed by 59% and 56%,respectively ,and the survival of C666-1 cells was reduced by 33% and 24% respectively at 48 h after siRNA1414 and siRNA1437 transfection. PCR-microarray analysis revealed that arachidonate 12-lipoxygenase （ALOX12） protein expression was down-regulated and glutathione reductase （GSR） and peroxiredoxin 3 （PRDX3） expression were transcriptionally up-regulated at 48 h after the suppression of EBNA1 expression in C666-1 cells. 【Conclusions】 EBNA1 maybe contribute to the oncogenesis of nasopharyngeal carcinoma （NPC） indirectly by up-regulating ALOX12 expression.

关 键 词：EB病毒 EB病毒核抗原1 RNA干扰 鼻咽癌 

分 类 号：R512.62[医药卫生—内科学]