山东大学耳鼻喉眼学报 ›› 2015, Vol. 29 ›› Issue (3): 51-53.doi: 10.6040/j.issn.1673-3770.0.2014.381

• 论著 • 上一篇    下一篇

靶向人喉癌HEP2细胞Trop2基因的siRNA构建及鉴定

陈小玲1,2, 费兵1,3, 李贤斌2, 吴昊1   

  1. 1. 南通大学附属医院耳鼻咽喉头颈外科, 江苏 南通 226001;
    2. 枣阳市第一人民医院耳鼻喉科, 湖北 枣阳 441200;
    3. 金湖县人民医院耳鼻喉科, 江苏 金湖 211600
  • 收稿日期:2014-12-02 修回日期:2015-04-03 发布日期:2015-06-16
  • 通讯作者: 吴昊。E-mail:entwuhao@163.com E-mail:entwuhao@163.com
  • 作者简介:陈小玲。E-mail:chenxiaoling-321@163.com

Construction and identification of small interfering RNA targeting silence Trop2 expression in human laryngeal carcinoma cell line HEP2

CHEN Xiaoling1,2, FEI Bing1,3, LI Xianbin2, WU Hao1   

  1. 1. Department of Otolaryngology & Head and Neck Surgery, Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu, China;
    2. Department of Otolaryngology, Zaoyang First People's Hospital, Zaoyang 441200, Hubei, China;
    3. Department of Otolaryngology, Jinhu People's Hospital, Jinhu 211600, Jiangsu, China
  • Received:2014-12-02 Revised:2015-04-03 Published:2015-06-16

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摘要： 目的 构建能高效干扰Trop2基因的siRNA, 转染人喉癌HEP2细胞, 并初步鉴定其干扰效果。方法 以人Trop2基因为靶基因, 设计合成3条针对不同作用位点的siRNA干扰序列, 用脂质体法将各小干扰序列瞬时转染人喉癌HEP2细胞, 同时设立阴性对照、空白对照, 分别命名为W组(未转染组)、NC组(阴性对照组)、T1组(S1序列组)、T2组(S2序列组)及T3组(S3序列组), 每组各5例。荧光显微镜下观察绿色荧光蛋白表达, 计算转染效率。转染24 h后, 收集稳定后细胞, 采用实时荧光定量PCR法检测各干扰序列对Trop2基因表达的抑制效果。结果 荧光显微镜下观察, 见HEP2细胞表达绿色荧光蛋白, 证实siRNA已转入细胞, 转染率达90%。RT-PCR法结果显示, 与未转染组、阴性对照组相比, 转染siRNA的人喉癌HEP2细胞中Trop2表达均受到抑制, 与T2、T3组相比, T1组对Trop2 mRNA的抑制作用最明显, 差异有统计学意义(0.470±0.063 vs 0.749±0.024, 0.824±0.027, P<0.05)。结论 成功构建并筛选出了能高效、特异沉默人喉癌HEP2细胞Trop2基因的siRNA序列, 为进一步研究Trop2基因在喉癌中的作用机制及其基因治疗奠定了基础。

关键词: Trop2, HEP2细胞, 喉肿瘤, RNA干扰

Abstract: Objective To construct the small interfering RNA(siRNA) that can silence Trop2 expression and to investigate the silencing effect of siRNA on Trop2 gene in human laryngeal carcinoma cell line HEP2. Methods Three different gene sequences siRNA specific targeting Trop2 gene were constructed and used to transfect into HEP2 cells by using LipofectamineTM 2000. The negative control group and blank control group were set up at the same time. These cells were divided into the W group (un-transfection), the NC group (transfection with negative control), the T1 group(S1 sequence), the T2 group(S2 sequence) and the T3 group(S3 sequence). The transfection efficiency was reported by green fluorescence protein expression and gene inhibition efficiency was analyzed by real-time RT-PCR. Results Green fluorescence observed by fluorescence microscopy showed that siRNA had been transfected into HEP2 cells, and the transfection ratio was 90%. The expression levels of Trop2 mRNA in cells transfected with Trop2-specific siRNA were lower than those in the untransfected cells or cells transfected with negative control siRNA. Compared with the T2 and T3 groups, siRNA in the T1 group had the strongest inhibitory effect on the expression of Trop2 mRNA, and the difference was significant(0.470±0.063 vs 0.749±0.024, 0.824±0.027, P<0.05 ). Conclusion Effective siRNA sequence has been successfully constructed and selected which can inhibit the expression of Trop2 gene in human laryngeal carcinoma cell line HEP2. It is fundamental for the study on the function of Trop2 gene and the gene therapy of laryngeal carcinoma.

Key words: HEP2 cells, Laryngeal carcinoma, RNA interference, Trop2

中图分类号: 

  • R739.6
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