转染沉默IGF1R基因的肝癌细胞株增殖、迁移及侵袭能力观察  

Construction of RNAi lentiviral vector targeting IGF1R gene and its effect on proliferation,migration and invasion of hepatocellular carcinoma cells

作　　者：别彩群[1] 黄秋燕[2] 颜英[2] 石珩[2] 汤绍辉[2] 

机构地区：[1]广州医科大学附属深圳沙井医院,广东深圳518104 [2]暨南大学附属第一医院

出　　处：《山东医药》2016年第11期1-4,共4页Shandong Medical Journal

基　　金：深圳市科技计划研究项目(JCYJ20140414160300592)

摘　　要：目的设计并筛选高效沉默胰岛素样生长因子1受体(IGF1R)基因的小干扰RNA(siRNA),构建其慢病毒表达载体,观察转染沉默IGF1R基因的肝癌细胞株增殖、迁移及侵袭能力。方法根据siRNA设计原则,参照IGF1R mRNA序列设计3对siRNA序列及1对阴性对照序列,转染肝癌细胞株Huh7 24 h后采用实时荧光定量PCR检测IGF1R mRNA,筛选干扰效率最高的siRNA序列。构建该序列的慢病毒表达载体,转染293T细胞进行病毒包装。将包装好的慢病毒表达载体感染肝癌细胞株Huh7和Hep3B,筛选沉默IGF1R基因表达的稳定细胞株。将上述稳定细胞株扩大培养,观察细胞IGF1R mRNA表达变化及细胞增殖、迁移与侵袭能力变化。结果实时荧光定量PCR显示,IGF1R_002序列在100 nmol/L浓度时抑制效率最高,达78.6%。与未感染任何病毒的Huh7、Hep3B细胞,感染p LVX-shRNA2空载体的Huh7、Hep38细胞相比,感染携带IGF1R干扰序列慢病毒的Huh7、Hep3B细胞IGF1R mRNA表达水平显著下调,细胞增殖活性明显降低,细胞迁移和侵袭能力明显受到抑制(P均<0.01)。结论筛选出1对高效沉默IGF1R基因的siRNA序列,即IGF1R_002;该siRNA介导的IGF1R基因沉默可明显抑制肝癌细胞株Huh7和Hep3B增殖、迁移与侵袭。Objective To design and screen an effective small interfering RNA（ siRNA） targeting human insulin-like growth factor 1 receptor（ IGF1R） gene,to construct the siRNA lentiviral vector,and to observe its effect on the proliferation,migration and invasion of hepatocellular carcinoma cells. Methods According to siRNA design principle,three siRNAs targeting IGF1 R gene and one negative control siRNA were designed and synthesized. They were transfected into Huh7 cells. IGF1 R mRNA expression levels in Huh7 cells were detected by real-time fluorescent quantitative PCR at 24 h after transfection,and we screened the most effective siRNA. After lentiviral expression vector was constructed,it was transfected into 293 T cells and packed into lentiviral. Huh7 and Hep3 B cells were infected with the p LVX-shRNA2-IGF1R_002lentiviral to screen the stable cell lines. The stable cell lines were cultured in large. We detected the changes in the IGF1 R mRNA expression,cell proliferation,cell migration and invasion. Results The real-time fluorescent quantitative PCR showed that IGF1R_002 showed the highest inhibition effect（ relative inhibition rate 78. 6%） at the concentration of 100 nmol / L. The expression levels of IGF1 R mRNA in Huh7 and Hep3 B cells with the lentiviral were significantly reduced,the proliferation abilities were inhibited,and the abilities of cell migration and invasion were significantly decreased as compared with those of Huh7 and Hep3 B cells without any virus infection and Huh7 and Hep3 B cells infected with p LVX-shRNA2 empty vector（ all P〈0. 01）. Conclusions The most effective siRNA targeting human IGF1 R gene,namely IGF1R_002,is screened out. The most effective siRNA-mediated IGF1 R gene silencing can significantly suppress the proliferation,migration and invasion of Huh7 and Hep3 B cells.

关 键 词：RNA干扰 人胰岛素样生长因子1受体基因 小干扰RNA 肝细胞癌 

分 类 号：R735.7[医药卫生—肿瘤]