shRNA介导胰岛素样生长因子Ⅰ类受体基因沉默诱导人肺癌A549细胞凋亡的体内外研究

shRNA-mediated insulin-like growth factor I receptor gene silencing inhibits cell proliferation,induces cell apoptosis,and suppresses tumor growth in non-small cell lung cancer:in vitro and in vivo experiments

目的评价 RNA 干扰(RNA interference,RNAi)技术对人肺癌 A549细胞系中胰岛素样生长因子Ⅰ类受体(IGF-IR)表达的抑制作用及其体内外抑瘤效应。方法将 IGF-IR 特异 shRNA转染人肺癌 A549细胞株,检测 IGF-IR 表达水平和 bcl-2及 caspase-3的表达变化;MTT 法检测体外培养细胞的生长抑制率;流式细胞技术检测细胞增殖周期;Western 印迹法分析 Akt 和 ERK 磷酸化程度;荷瘤裸鼠肺癌模型中观察瘤内注射的抑瘤效应及凋亡情况。结果 IGF-IR 表达抑制率达89.8%,bcl-2表达为对照组的46%±6%,caspase-3激活为对照组的156%±8%,Akt、ERK 磷酸化分别为对照组的20%和36%±3%;肿瘤细胞活力明显下降;直接瘤内注射后肿瘤体积减少至对照组的40%～50%,并促进肿瘤细胞凋亡。结论运用 RNAi 技术能有效抑制 A549细胞 IGF-IR 的表达,使细胞增殖能力减弱,凋亡增加,瘤体生长受抑。

Objective To study the effects of RNA interference （RNAi）-mediated insulin-like growth factor I receptor （IGF-IR） gene silencing on human lung cancer cells. Methods Plasmids expressing IGF-IR shRNA1 and IGF-IR shRNA2 were constructed. Human non-small cell lung cancer cells of the line A549 were cultured and transfected with sequence-specific shRNA. RT-PCR was used to monitor the IGF-IR mRNA expression. Western blotting was used to detect the expression of IGF-IR, bcl-2 and caspase-3, associated with apoptosis, and IGF-IR signaling pathways-associated proteins, total and phospho- ERK1/2 and Akt. MTr assay and flow cytometry were used to examine the cell activity and cell cycle. Twelve nude mice were injected subcutaneously with A549 cells, 20 days later the mice were randomly divided into 3 groups to be injected into the tumor with IGF-IR, PBS, or blank plasmid respectively 4 times with the interval of 5 days. Five days after the 4th injection the mice were killed and the tumors were taken out. TUNNEL assay was used to detect the apoptotic cell in the tumor. Results RT-PCR showed that the IGF-IR mRNA expression level of the A549 cells transfected with IGF-IR shRNA1 was only 24% ± 4% that of the A549 cells transfected with blank plasmid （P 〈 0.05） ; however, the IGF-IR mRNA expression level of the A549 cells transfected with IGF-IR shRNA2 was 78% ± 5% that of the A549 cells transfected with blank plasmid （P 〉 0. 05）. The IGF-IR protein expression level of the A549 cells of the IGF-IR shRNA1 group was only 10. 2% ± 2.8% that of the A549 cells of the blank plasmid group （ P 〈 0. 05 ）. Western blotting showed that the protein expression levels of bcl-2 and caspace-3p20 of the A549 cells of the IGF-IR shRNA1 group were 46% ± 6% and 156% ± 8% those of the negative controls （ both P 〈 0.05 ） ; however, the protein expression levels of bcl-2 and caspace-3p20 of the A549 cells of the IGF-IR shRNA2 group were not different from those of the negative control cells. The Akt kinase and ERK phosphorylation levels of the A549 cells of the IGF-IR shRNA1 group were 10% and 36% ±3% those of the negative control cells respectively （both P 〈 0.05）. Since 48h after the transfection the active cell number of the IGF-IR shRNA1 group was 64% ± 7% that of the negative group （ P 〈 0.05）, and this decrease effect lasted to 72 h after （67% ± 6% that of the negative cells, P 〈 0.05 ）. 48 h after the transfection the percentage of cells at G0/ G1 phase of the IGF-IR shRNA1 group was 77.5%, significantly higher than that of the negative control group, and the percentages of the cells at S and G2/M phases of the IGF-IR shRNA1 group were 15.7% and 7.3% respectively, both significantly lower than those of the negative control group （23.0% and 29.9% respectively）. Since the second injection the tumor size of the mice of IGF-IR shRNA group was 40% -50% that of the PBS group （P 〈 0.05 ）, and the tumor size of the mice of the PBS group was 90% that of the control group. TUNNEL assay showed that the number of apoptotic cells in the tumors of the IGF-IR shRNA1 group mice was 118 ± 8/high power, significantly higher than that of the control group （70 ± 9, P 〈 0.05 ）. Conclusion RNAi technique effectively inhibits the expression of IGF-IR, thus decreasing the NSCLC cell proliferation inducing apoptosis and inhibiting the tumor growth.