核糖核酸干扰缺氧诱导因子-1α对食管鳞癌细胞的抑制效果研究  

In vitro and in vivo inhibitory effect of RNA interference targeting hypoxia-inducible factor-lα in esophageal squamous cancer cells

作　　者：张捷[1] 施瑞华[1] 肖斌[1] 张国新[1] 郝波[1] 

机构地区：[1]南京医科大学第一附属医院消化科,210029

出　　处：《中华消化杂志》2008年第1期26-29,共4页Chinese Journal of Digestion

基　　金：江苏省自然科学基会资助项目（BK2005153）

摘　　要：目的探讨人食管鳞癌细胞系Eca-109中RNA干扰抑制缺氧诱导因子（HIF）-1α的体内、外实验效果。方法用针对HIF-1α mRNA的小发夹RNA（shRNA）真核表达载体转染人食管鳞癌细胞Eca-109，检测HIF-1α、基质金属蛋白酶（MMP）-2、葡萄糖载体蛋白（GLUT）-1表达，挑选干扰效果最好的细胞命名为Eca-109／shRNA，流式细胞仪检测细胞凋亡率。6周龄BALB／C裸鼠30只，按皮下注入细胞不同均分为三组，A组为Eca-109，B组为转染空质粒Eca-109（Eca-109／Neo），C组为Eca-109／shRNA。观察肿瘤出现时间，测量裸鼠体质量、瘤体大小，计算肿瘤体积。28d后处死全部裸鼠，取瘤体称重。用Western印迹法检测肿瘤组织HIF-1α蛋白、血管内皮生长因子（VEGF）、凋亡抑制蛋白bcl-2和生存素的表达。结果Eca-109／shRNA细胞的HIF-1α蛋白表达明显被抑制，同时MMP-2，GLUT-1表达也明显下调，细胞凋亡率为（21．32±1．12）％，比Eca-109细胞和Eca-109／Neo细胞[（1．17±0．85）％和（5．31±1．46）％]明显升高（P〈0．01）。A、B组裸鼠接种1周左右成瘤，C组裸鼠2周左右成瘤，成瘤时间较A、B组延迟，且瘤体生长减慢。28d后移植瘤体积：A组为（1．29士0．34）cm^3，B组为（1．19±0．35）cm^3，C组为（0．45士0．20）cm^3。瘤重：A组为（0．73±0．13）g，B组为（0．71士0．15）g，C组为（0．21±0．11）g。C组与A、B组比较移植瘤体积和瘤重差异均有统计学意义（P值均〈0．01），抑瘤率达65％。Western印迹检测蛋白表达，C组HIF-lα、VEGF、bcl-2和生存素表达均比A、B组明显减少（P值均〈0．01）。结论在食管癌Eca-109细胞中利用RNA干扰HIF-1α能抑制肿瘤生长，其机制为抑制VEGF介导的肿瘤血管生成和无氧糖酵解，可能与影响bcl-2和生存素的表达，促进肿瘤细胞凋亡有关。Objective To investigate the in vitro and in vivo effect of RNA intederence targeting hypoxiainducible factor-lα（HIF-lα）in human esophageal squamous cancer cell line Eca-109. Methods Human esophageal squamous cancer Eca-109 cells were transfected with small hairpin RNA（shRNA） eukaryotic expression vector targeting HIF-lα gene. The Eca-109 cells which stably inhibited by shRNA were named Eca-109/shRNA. The expressions of HIF-lα, MMP-2 and GLUT-1 were evaluated. The apoptosis was determined by flow cytometric analysis. BALB/C nude mice （6 weeks old） were injected with either Eca-109 cells （n = 10）, or Eca-109 cells transfected with empty expression vector（Eca-109/Noe） （n = 10）, or Eca-109 cells stable transfected with shRNA（Eca-109/shRNA） （n = 10）. The tumors formation, the body weight and the volume of the tumor were measured. The HIF-lα, VEGF, bcl-2, survivin expression in tumor tissues were determined by Western blot. Results The expression of HIF-lα were significantly lower in transfected Eca-109 cells （Eca-109/shRNA）. The apoptosis was increased in Eca-109/shRNAE（21.32 ± 1.12） % ] as compared to Eca-109E（1.17 ± 0. 85） %] and Eca-109/ Neon（5. 31 ± 1. 46）%] （ P 〈 0.01）. In vivo, the tumor formation was 2 weeks in Eca-109/shRNA injected mice, which was longer than those injected with Eca-109 and Eca-109/Noe（1 week）. The volume and the weight of tumor in Eca-109/shRNA injected mice[-（0. 45 ± 0. 20）cm^3 and（0. 21 ±0. ll）g, respectively]were significantly lower than those injected with Eca-109[-（1.29±0.34）cm^3 and （0.73 ± 0. 13）g, respectively] and Eca-109/Neo 1-（ 1.19 ± 0.35）cm^3 and （0.71± 0.15）g, respectively],（P （0.01）. The inhibition rate of tumor was 65%. The expressions of HIF-lα, VEGF, bcl-2 and survivin were significantly lower in Eca-109/shRNA injected mice than those injected with Eca-109 and Eca-109/Noe （P〈 0.01）. Conclusions shRNA mediated-disruption of HIF-la suppresses tumor growth in vivo, wh

关 键 词：食管癌 RNA 小分子干扰 缺氧诱导因子-1Α 

分 类 号：R686[医药卫生—骨科学]