靶向RNAi体外沉默IGF-IR基因后非小细胞肺癌A549细胞的生物学行为及化学敏感性

[Biological behaviors and chemosensitivity of NSCLC A549 cells after IGF-IR gene silencing by targeting RNAi in vitro].

作者信息

Kong Min-jian, Dong Ai-qiang, Wu Wei, Ma Zhi-yuan, Cheng Hai-feng, Qian Jian-fang, Fan Jun-qiang

机构信息

Department of Cardiothoracic Surgery, The Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310009, China.

出版信息

Zhejiang Da Xue Xue Bao Yi Xue Ban. 2008 Jul;37(4):373-80. doi: 10.3785/j.issn.1008-9292.2008.04.009.

DOI:10.3785/j.issn.1008-9292.2008.04.009

PMID:18705010

Abstract

OBJECTIVE

To investigate the biological behaviors and chemosensitivity of non-small cell lung cancer (NSCLC) cell line A549 after IGF-IR gene silencing by RNA interference (RNAi) in vitro.

METHODS

Two plasmids siRNA 1 and 2 expressing IGF-IR siRNA with human U6 promoter were constructed,and an unrelated siRNA was used as negative control. NSCLC A549 cells were transfected with sequence-specific siRNA or unrelated siRNA as control. Quantitative RT-PCR and Western blot were used to detect the expression of IGF-IR. NSCLC A549 cells were transfected with siRNA and treated with DDP. MTT assay and flow cytometry were used to assess the effects of IGF-IR silencing on tumor cell proliferation and chemosensitivity.

RESULT

Transfection of NSCLC cells with siRNA resulted in reduction of IGF-IR mRNA expression by 78.9 % and protein production by 89.8%. The decrease in IGF-IR levels caused significant growth inhibition of A549 cells both at 48 h and at 72 h, and decrease of the IC50 of DDP at 24 h, 48 h and at 72 h. Flow cytometry showed that 77.5% of A549 cells retained in G0/G1 phase.

CONCLUSION

The sequence specific suppression of IGF-IR gene expression by RNAi enhances sensitivity to DDP in NSCLC cell.

摘要

目的

在体外研究RNA干扰（RNAi）沉默IGF-IR基因后非小细胞肺癌（NSCLC）细胞系A549的生物学行为和化学敏感性。

方法

构建两种带有人类U6启动子表达IGF-IR siRNA的质粒siRNA 1和2，并使用无关的siRNA作为阴性对照。用序列特异性siRNA或无关siRNA作为对照转染NSCLC A549细胞。采用定量RT-PCR和蛋白质免疫印迹法检测IGF-IR的表达。用siRNA转染NSCLC A549细胞并给予顺铂（DDP）处理。采用MTT法和流式细胞术评估IGF-IR沉默对肿瘤细胞增殖和化学敏感性的影响。

结果

用siRNA转染NSCLC细胞导致IGF-IR mRNA表达降低78.9%，蛋白质生成减少89.8%。IGF-IR水平的降低在48小时和72小时均导致A549细胞显著生长抑制，并在24小时、48小时和72小时导致DDP的半数抑制浓度（IC50）降低。流式细胞术显示77.5%的A549细胞停滞于G0/G1期。