小干扰RNA沉默cox-2表达增强食管癌EC9706细胞的放射敏感性  

Small interfering RNA in silencing cox-2 expression enhances radiosensitivity of esophageal cancer EC9706 cell

作　　者：吴慧[1] 卢晓旭 刘冬梅 赵国强[3] 樊青霞[1] 

机构地区：[1]郑州大学第一附属医院肿瘤科,450052 [2]郑州大学附属肿瘤医院放疗科 [3]郑州大学基础医学院

出　　处：《中华医学杂志》2012年第23期1621-1625,共5页National Medical Journal of China

摘　　要：目的探讨环氧化酶（cox-2）基因的特异性小干扰RNA（siRNA）对食管癌EC9706细胞的放射增敏作用及机制。方法构建针对cox-2基因的siRNA载体，通过脂质体转染技术将其转入食管癌EC9706细胞，G418筛选得到稳定转染细胞株。0、2、4Gy辐射处理，荧光定量RT-PCR检测细胞中cox-2、MMP2、Bax、Bcl-2的表达水平；Western印迹检测Cox-2蛋白及AKT蛋白和磷酸化AKT蛋白（pAKT）的表达；流式细胞仪检测细胞凋亡；细胞体外侵袭实验检测细胞侵袭能力；克隆形成实验检测不同辐射处理对细胞增殖能力的影响。结果测序证实构建出针对cox-2基因的siRNA载体pRNA—U6．1-siCox214，RT—PCR和Western印迹结果显示其转染的食管癌EC9706细胞cox-2基因表达为有效沉默。0、2、4Gy辐射处理后，沉默cox-2基因表达的EC9706细胞中MMP2、Bcl-2mRNA、AKT蛋白和磷酸化AKT表达水平均显著降低，且与照射剂量呈反比，BaxmRNA表达显著升高，且与照射剂量呈正比；cox-2沉默组细胞凋亡率明显高于对照组，差异有统计学意义（P〈0．01）；cox-2沉默组细胞外侵袭穿透细胞数显著减少，差异有统计学意义（P〈0．01）。cox-2沉默组细胞在0、1、2、4、6、8、10Gy照射后集落形成率均显著降低（P〈0．01）。结论siRNA沉默cox-2基因表达可明显增强食管癌EC9706细胞的放射敏感性，可能与MMP2、Bax、Bcl-2、AKT蛋白和磷酸化AKT蛋白有关。Objective To explore the effects of small interfering RNA （siRNA） specific to cox-2 gene on the radiosensitivity of esophageal cancer cell EC9706. Methods The siRNA vector was established for cox-2 gene and then induced into esophageal cancer cell EC9706 by lipofectamine. G418 screening yielded stably transfected cells. After the irradiation of 0, 2 and 4 Gy, the cellular expression levels of cox- 2, matrix metalloproteinase-2 （MMP2）, Bax and Bcl-2 were detected by reverse transcriptase-polymerase chain reaction （RT-PCR） and those of cox-2 protein, AKT protein and phosphorylation AKT protein （pAKT） by Western blot. Cell apoptosis was examined by flow cytometer. Invasion of cells was detected by invasive assay in vitro. The invasive and metastatic capacities of cancer cells were assessed by invasion assay in vitro. Proliferative potential was quantified by clone-forming assay. Results The sequencing result confirmed that siRNA vector pRNA-U6 for cox-2 gene was established. The results of 1-sinCox214, RT-PCR and Western blot showed that cox-2 gene expression of transfected EC9706 cell was silenced efficiently. After the irradiation of 0, 2 and 4 Gy, the expressions of MMP2, Bcl-2 mRNA, AKT protein and pAKT in silencing cox-2 gene expression significantly decreased. There was an inverse correlation with irradiation dose. The Bax mRNA expression evidently increased directly with irradiation dose; the apoptotic rate in cox- 2 silencing groups was evidently higher than the control groups. And the difference was significant （P 〈 0. 01 ） ; invasion cells in vitro in Cox-2 silencing groups evidently decreased with significant difference （P 〈 0. 01 ）. The colony formation rate of cells decreased obviously in cox-2 silencing groups after the irradiation of 0, 1,2, 4, 6, 8 and 10 Gy （P 〈 0. 01 ）. Conclusions Small interference RNA in silencing cox-2 gene expression can enhance significantly the radiosensitivity of esophageal cancer EC9706 cells. And themechanism may be related with MMP2, Bax

关 键 词：食管肿瘤 RNA 小分子干扰 辐射耐受性 环氧合酶2 

分 类 号：R735.1[医药卫生—肿瘤]