RNAi介导的IGF1R基因沉默对肝癌细胞生长、迁移与侵袭的影响

Effect of RNAi-mediated IGF1R gene silencing on growth,migration,and invasion of hepatocellular carcinoma cells

目的:研究RNA干扰(RNAi)介导的胰岛素样生长因子1受体(IGF1R)基因沉默对肝癌细胞生长、迁移与侵袭的影响。方法:设计并筛选抑制IGF1R mRNA表达效率最高的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。将包装好的慢病毒感染Huh7和Hep3B肝癌细胞,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,检测细胞IGF1R mRNA表达变化,细胞生长、迁移与侵袭能力变化,以及Ki-67、p-AKT、p-ERK1、Gli1、β-catenin、cyclin D1、p21、BCL-XL的蛋白表达水平变化。结果:与空白及阴性对照组比较,感染携带IGF1R干扰序列慢病毒的Huh7和Hep3B肝癌细胞IGF1R mRNA表达水平显著下调,细胞增殖活性明显降低,细胞凋亡显著增加,细胞迁移和侵袭能力明显受到抑制,细胞中IGF1R、Ki-67、p-AKT、p-ERK1、Gli1、β-catenin、cyclin D1、p21及BCL-XL蛋白表达水平均显著降低。结论:RNAi介导的IGF1R基因沉默可明显抑制Huh7和Hep3B肝癌细胞生长及恶性生物学特征,这可能与IGF1R表达水平显著下调而引起上述调控细胞增殖、抗凋亡基因以及相关信号通路基因的蛋白表达水平显著降低有关。

AIM： To investigate the effect of RNA interference（ RNAi）-mediated insulin-like growth factor 1receptor（ IGF1R） gene silencing on the growth,migration,and invasion of hepatocellular carcinoma cells. METHODS：The most effective siRNA targeting IGF1 R gene was designed and screened. After lentiviral expression vector p LVX-shRNA2-IGF1 R carrying the most effective siRNA sequence was constructed,it was transfected into 293 T cells and packed into p LVX-shRNA2-IGF1 R lentivirus. Huh7 and Hep3 B cells were infected with the p LVX-shRNA2-IGF1 R lentivirus to screen the positive clone Huh7 cells and Hep3 B cells with the lentivirus. These Huh7 cells and Hep3 B cells were cultured to analyze the mRNA level of IGF1 R,cell proliferation,cell cycle,cell apoptosis,cell migration / invasion,and the protein levels of IGF1 R,Ki-67,p-AKT,p-ERK1,Gli1,β-catenin,cyclin D1,p21 and BCL-XL. RESULTS： The mRNA expression of IGF1 R in Huh7 cells and Hep3 B cells with p LVX-shRNA2-IGF1 R lentivirus was significantly reduced. The proliferation of these cells was remarkably inhibited,and the number in G1 phase was increased significantly. The percentages of apoptotic cells were increased markedly,and the number of cell migration / invasion was decreased markedly. The protein levels of IGF1 R,Ki-67,p-AKT,p-ERK1,Gli1,β-catenin,cyclin D1,p21 and BCL-XL were decreased significantly compared with the blank control group and negative control group. CONCLUSION： The RNAi-mediated IGF1 R gene silencing significantly suppresses the growth and the malignant biological characteristics of Huh7 cells and Hep3 B cells,which may be involved in the reduced protein levels of the above genes induced by down-regulation of IGF1 R expression.