Suppression of VEGF transcription in renal cell carcinoma cells using pyrrole-imidazole hairpin polyamides targeting the hypoxia responsive element

低氧诱导因子（Hypoxia inducible factor，HIF）是低氧环境下细胞存活的关键基因调控因子，与肾癌的发生、发展密切相关。N-甲基吡咯（Py）-N-甲基咪唑（Im）发夹型聚酰胺是一种人工合成的有机化合物，它能识别并结合特定DNA序列的小沟。我们针对血管内皮生长因子（VEGF）基因启动子区缺氧反应元件（HIF的结合位点）的侧翼序列合成了三种Py-Im发夹结构的聚酰胺。使用含有VEGF启动子的报告质粒通过荧光素酶测定来评价聚酰胺对HIF诱导的转录的影响。采用真实的实时逆转录聚合酶链反应（RT-PCR）和酶联免疫吸附试验（ELISA）检测聚酰胺对VonHippel-Lindau基因移码突变的A498肾癌细胞VEGF转录和分泌的影响。在使用293细胞的报告基因测定中，三种Py-Im发夹聚酰胺的组合抑制了HIF诱导的转录，并成功地抑制了A498细胞中VEGF基因的转录和翻译。使用Py-Im发夹聚酰胺的方法可能为治疗对常规化疗或放疗耐药的肾细胞癌提供新的策略。

Hypoxia inducible factor (HIF), a master regulator of critical genes for cell survival under hypoxia, is known to be related to tumorigenesis and progression of renal cell carcinoma. N-methylpyrrole (Py)-N-methylimidazole (Im) hairpin polyamides are synthetic organic compounds which recognize and bd to minor groove of specific DNA sequences. We synthesized three Py-Im hairpin polyamides targeting the flanking sequences of hypoxia responsive element (a binding site of HIF) in the promoter region of the vascular endothelial growth factor (VEGF) gene. Effects of the polyamides on HIF-induced transcription was evaluated by luciferase assay using a reporter plasmid containing VEGF promoter. Real time reverse transcriptase polymerase chain reaction and enzyme-linked immunosolvent assay were performed to examine effects of the polyamides on transcription and secretion of VEGF in A498 renal cell carcinoma cells which has a frame shift mutation in the von Hippel-Lindau gene. Combinations of three Py-Im hairpin polyamides suppressed HIF-induced transcription in reporter assays using 293 cells, and successfully suppressed transcription and translation of the VEGF gene in A498 cells. The approach using Py-Im hairpin polyamides may provide a new strategy for the treatment of renal cell carcinoma that is resistant to conventional chemotherapy or irradiation.