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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类受体(IGF-Ⅰ R)表达的阻断效应,和IGF-Ⅰ R基因沉默后细胞增殖、凋亡等生物学特性及肿瘤细胞对化疗药物敏感性的改变。方法:应用U6启动子,介导DNA模板转录生成短发夹样RNA(shRNA),并转染人肺癌A549细胞株,从而产生IGF-Ⅰ R特异性小干扰RNA(siRNA),经RT-PCR和Western blot检测IGF-Ⅰ R表达的改变;联合应用化疗药物顺铂(DDP),通过MTT法和流式细胞技术等,观察细胞生长、细胞周期、细胞凋亡及DDP半数致死量(IC50)的变化。结果:IGF-Ⅰ R表达水平明显下降(抑制率高达89.8%),肿瘤细胞增殖能力明显减弱,细胞滞留于G0期的比例上升;DDP对肿瘤细胞24h、48h、72h的IC50均明显减少,IGF-Ⅰ R siRNA1组DDP作用72h的IC50为0.92 mg/L,明显低于control-siRNA组的3.77mg/L,0.5mg/L DDP联合IGF-Ⅰ R siRNA1作用48h后,A549细胞的生长抑制率达32.1%,明显高于control-siRNA组的18.9%,细胞凋亡率从27.8%上升至44.2%。结论:运用RNAi技术能有效抑制A549细胞IGF-Ⅰ R的表达,使细胞增殖能力减弱,化疗敏感性增加。
OBJECTIVE: To evaluate the effect of RNA interference (RNAi) technology on blocking the expression of insulin-like growth factor receptor (IGF-ΙR) in human lung cancer cell line A549 and the effect of IGF-ΙR gene silencing on cell proliferation, Apoptosis and other biological characteristics and tumor cell chemosensitivity changes. Methods: U6 promoter was used to mediate the transcription of short hairpin RNA (shRNA) into human lung adenocarcinoma cell line A549 by RNAi mediated gene transfer to produce IGF-ⅠR-specific small interfering RNA (siRNA) And Western blot were used to detect the changes of IGF-ⅠR expression. The cell growth, cell cycle, apoptosis and DDP (IC50) were observed by MTT assay and flow cytometry (DDP) The change. Results: The expression of IGF-ⅠR was significantly decreased (the inhibition rate was as high as 89.8%). The proliferation of tumor cells was significantly weakened and the proportion of cells retained in G0 phase increased. The IC50 of DDP for 24h, 48h and 72h was significantly decreased -I R siRNA1 group DDP 72h IC50 0.92 mg / L, was significantly lower than the control-siRNA group 3.77mg / L, 0.5mg / L DDP combined with IGF-Ⅰ R siRNA1 effect 48h after A549 cell growth inhibition rate Reaching 32.1%, which was significantly higher than that of control-siRNA group (18.9%). The rate of apoptosis increased from 27.8% to 44.2%. Conclusion: The use of RNAi technology can effectively inhibit the expression of IGF-Ⅰ R in A549 cells, weakening the cell proliferation and increasing the chemosensitivity.