TRAF4沉默对食管癌细胞Eca-109增殖、凋亡及细胞周期的影响  

Effects of TRAF4 Silencing on the Proliferation,Apoptosis and Cell Cycle of Esophageal Cancer Cell Line Eca-109

作　　者：蒋鸣[1] 翟祥军[2] 徐池[1] 周国仁[1] 

机构地区：[1]江苏省肿瘤医院放疗科,南京210009 [2]江苏省疾病预防与控制中心,南京210009

出　　处：《华中科技大学学报（医学版）》2016年第4期389-393,401,共6页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

摘　　要：目的探讨小干扰RNA（siRNA）靶向沉默肿瘤坏死因子受体相关因子4（TRAF4）基因表达对人食管癌细胞恶性行为的影响。方法选取对数生长期Eca-109细胞并行脂质体法高效转染2条靶向沉默TRAF4的siRNA（siTRAF4-1、siTRAF4-2）,采用实时定量PCR检测转染后的TRAF4 mRNA水平以筛选沉默效果较好的siRNA为沉默组,同时设阴性对照组（转染阴性siRNA序列）和空白对照组。采用噻唑蓝（MTT）法检测转染24、48、72、96h的吸光度值以计算各组增殖抑制率,AnnexinⅤ-FITC/PI双染、Caspase-3FITC单染或PI单染流式细胞术检测转染后的凋亡率、Caspase-3活化率和细胞周期分布情况。结果靶向沉默TRAF4 24-96h内,转染siTRAF4-1、siTRAF4-2的Eca-109细胞中TRAF4mRNA水平均低于未转染或转染对照siRNA的细胞,且转染siTRAF4-1、siTRAF4-2后的沉默率均高于转染对照siRNA的细胞（均P〈0.05）;后续试验选取沉默率较高的siTRAF4-1进行研究。与空白对照组和阴性对照组相比,沉默组的增殖抑制率、凋亡率及Caspase-3活化率均升高,差异有统计学意义（均P〈0.05）;沉默组转染96h的G0/G1期细胞比例高于空白对照组和阴性对照组,而S、G2/M期细胞比例低于其余两组,以上差异均有统计学意义（均P〈0.05）;空白对照组和阴性对照组以上指标的差异均无统计学意义（均P〉0.05）。结论通过siRNA靶向沉默TRAF4基因表达的效果较好,可抑制细胞增殖并诱导凋亡、Caspase-3活化和细胞周期G0/G1期阻滞,为食管癌靶向治疗提供了方法学基础。Objective To investigate the effects of small interfering RNA（siRNA）targeted silencing of tumor necrosis factor receptor associated factor 4（TRAF4）gene on the malignant behaviors of human esophageal cancer cells. Methods Eca-109 cells at the logarithmic growth phase were transfected with 2 siRNAs targeting TRAF4（siTRAF4-1and siTRAF4-2）by using liposomes.Real-time quantitative PCR was used to detect the TRAF4 mRNA level after transfection in order to screen out the siRNA with better silencing effect.Besides,the negative control group（cells transfected with negative siRNA sequence）and blank control group were set up.The absorbance（A）value at 24,48,72 and 96hafter transfection were measured by MTT assay as to calculate the inhibition rate of cell proliferation.AnnexinⅤ-FITC/PI double staining,Caspase-3FITC single staining and PI single staining were used to detect the apoptosis rate,Caspase-3activation rate and cell cycle distribution,respectively.Results In Eca-109 cells transfected with siTRAF4-1or siTRAF4-2,TRAF4 mRNA levels were much lower than those without transfection or transfected with control siRNA within 24-96 hpost transfection.The silencing rates of cells transfected with siTRAF4-1 or siTRAF4-2 were significantly higher than those transfected with control siRNA（all P〈0.05）.siTRAF4-1 with higher silencing rate was selected for the follow-up experiment.The proliferation inhibition rate,apoptosis rate and Caspase-3activation rate were increased in silencing group as compared with those in the blank control group and negative control group,and the differences were statistically significant（all P〈0.05）.Ninety-six hours after transfection,the proportion of cells at G0/G1 phase was found to be higher and the proportions of cells at S and G2/M phase to be lower in silencing group than those in blank control group and negative control group（all P〈0.05）.There were no significant differences between the control group and the negative control group in the aforementioned indic

关 键 词：小干扰RNA 肿瘤坏死因子受体相关因子4 食管癌细胞Eca-109 增殖 凋亡 

分 类 号：R735.1[医药卫生—肿瘤]