Department of Geriatrics, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China.
Mol Biol Rep. 2013 Apr;40(4):3093-9. doi: 10.1007/s11033-012-2383-4. Epub 2013 Mar 4.
It is reported that the autocrine loop of the vascular endothelial growth factor (VEGF) is crucial for the survival and proliferation of non-small cell lung cancer (NSCLC) tumors. In this study we aimed to systematically investigate the role of autocrine vascular VEGF in NSCLC cell line A549 through inhibition of endogenous VEGF. A549 cells were transfected with florescence-labeled VEGF oligodeoxynucleotide with lipofectamine. For the experimental group, cells were transfected with VEGF anti-sense oligodeoxynucleotide (ASODN), sense oligodeoxynucleotide (SODN) and mutant oligodeoxynuleotide (MODN) respectively. For the control group cells were mock transfected with lipofectamine or culture medium. At indicated time point after transfection, the expression levels of VEGF mRNA and protein in A549 cells were analyzed by RT-PCR and ELISA respectively. Cell viability was measured by the MTT assay. Cell cycle distribution was detected by flow cytometry. As revealed by RT-PCR assay, the mRNA level of VEGF in cells transfected with ASDON was significantly lower than the other four groups (P < 0.05) at 24 and 48 h after transfection. ELISA assay yielded similar result with significantly decreased level of VEGF protein expression (P < 0.05). The survival fraction of A549 cells transfected with ASDON was significantly lower than the other four groups (P < 0.05) at 24 h after transfection. Also the percentage of G2 phase cells of ASODN group was significantly lower than other four groups. Our data indicate that VEGF expression is efficiently inhibited in A549 cells by ASODN transfection and this inhibition leads to inhibited cell growth and impaired cell cycle distribution.
据报道，血管内皮生长因子（VEGF）的自分泌环对于非小细胞肺癌（NSCLC）肿瘤的存活和增殖至关重要。在这项研究中，我们旨在通过抑制内源性 VEGF 来系统地研究自分泌血管 VEGF 在 NSCLC 细胞系 A549 中的作用。使用脂质体将荧光标记的 VEGF 寡核苷酸转染到 A549 细胞中。对于实验组，分别用 VEGF 反义寡核苷酸（ASODN）、正义寡核苷酸（SODN）和突变寡核苷酸（MODN）转染细胞。对于对照组，细胞用脂质体或培养基模拟转染。转染后指定时间点，通过 RT-PCR 和 ELISA 分别分析 A549 细胞中 VEGF mRNA 和蛋白的表达水平。通过 MTT 测定法测量细胞活力。通过流式细胞术检测细胞周期分布。RT-PCR 检测结果显示，转染 ASDON 的细胞中 VEGF mRNA 的水平在转染后 24 和 48 小时显着低于其他四组（P <0.05）。ELISA 检测也得到了相似的结果，VEGF 蛋白表达水平显着降低（P <0.05）。转染 ASDON 的 A549 细胞的存活分数显着低于其他四组（P <0.05）。ASODN 组的 G2 期细胞百分比也显着低于其他四组。我们的数据表明，ASODN 转染可有效抑制 A549 细胞中 VEGF 的表达，从而抑制细胞生长和受损的细胞周期分布。
