转染沉默IGF1R基因的肝癌细胞株增殖、迁移及侵袭能力观察

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Huh724 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pLVX-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Hep3 B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 prolifera-tion, migration and invasion of hepatocellular carcinoma cells .Methods According to siRNA design principle , three siR-NAs targeting IGF1R gene and one negative control siRNA were designed and synthesized .They were transfected into Huh 7 cells.IGF1R 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 transfect-ed into 293T cells and packed into lentiviral .Huh7 and Hep3B cells were infected with the pLVX-shRNA2-IGF1R_002 lentiviral to screen the stable cell lines .The stable cell lines were cultured in large .We detected the changes in the IGF 1R 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 IGF1R mRNA in Huh7 and Hep3B cells with the lentiviral were significantly reduced , the proliferation abilities were inhibited , and the abilities of cell migration and invasion were significantly decreased as com-pared with those of Huh7 and Hep3B cells without any virus infection and Huh7 and Hep3B cells infected with pLVX-shR-NA2 empty vector (all P＜0.01).Conclusions The most effective siRNA targeting human IGF1R gene, namely IGF1R_002, is screened out.The most effective siRNA-mediated IGF1R gene silencing can significantly suppress the prolifera-tion, migration and invasion of Huh 7 and Hep3B cells.