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目的:研究小干扰RNA(small interfering RNA,siRNA)抑制Rac1b表达后对胃癌AGS细胞血管生成相关分子表达的影响。方法:体外合成针对Rac1b基因的siRNA序列(Rac1bsiRNA),脂质体法转染AGS细胞,RT-PCR和Western blotting观察Rac1b siRNA对AGS细胞Rac1b mRNA和蛋白水平表达的影响,ELISA和Western blotting检测缺氧条件下转染Rac1b siRNA后AGS细胞培养上清中VEGF表达水平以及细胞中血管生成相关分子P53、VHL和HIF-1α表达水平的变化。结果:测序证实体外合成的Rac1b siRNA序列正确。Rac1b siRNA转染AGS细胞后,可在mRNA和蛋白水平特异性抑制Rac1b的表达,对其同源分子Rac1的mRNA和蛋白表达水平无影响。Rac1bsiRNA可显著抑制AGS细胞培养上清中VEGF的分泌,这种抑制作用在缺氧情况下更为明显。同时,Rac1bsiRNA在缺氧情况下可抑制AGS细胞内HIF-1α蛋白的表达、上调p53和VHL蛋白的表达。结论:Rac1b siRNA可抑制胃癌AGS细胞中Rac1b在mRNA和蛋白水平的表达,可能通过调节血管生成相关分子HIF-1α、P53及VHL的表达抑制缺氧情况下AGS细胞VEGF的分泌。
AIM: To investigate the effect of small interfering RNA (siRNA) on the expression of angiogenesis-related molecules in gastric cancer AGS cells after inhibiting the expression of Rac1b. Methods: The Rac1b siRNA of Rac1b gene was synthesized in vitro and transfected into AGS cells by lipofectamine. The effect of Rac1b siRNA on the expression of Rac1b mRNA and protein in AGS cells was detected by RT-PCR and Western blotting. The levels of Rac1b mRNA and protein were detected by ELISA and Western blotting The expression of VEGF in supernatant of AGS cells and the expression of angiogenesis-related molecules P53, VHL and HIF-1α in AGS cells transfected with Rac1b siRNA under oxygen conditions. Results: Sequencing confirmed that the Rac1b siRNA sequence synthesized in vitro was correct. Rac1b siRNA transfected AGS cells could specifically inhibit the expression of Rac1b at mRNA and protein levels and had no effect on the mRNA and protein expression of its cognate molecule Rac1. Rac1bsiRNA can significantly inhibit VEGF secretion in AGS cell culture supernatants, which is more obvious in hypoxia. In the meantime, Rac1bsiRNA can inhibit the expression of HIF-1αprotein and upregulate the expression of p53 and VHL protein in AGS cells in hypoxia. CONCLUSION: Rac1b siRNA can inhibit the expression of Rac1b mRNA and protein in gastric cancer AGS cells. It may inhibit the secretion of VEGF in AGS cells by regulating the expression of angiogenesis-related molecules HIF-1α, P53 and VHL.