Rac1b siRNA对胃癌AGS细胞中血管生成相关分子表达的影响

Effect of Rac1b siRNA on expression of angiogenesis-related molecules in gastric cancer AGS cells

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。

Objective：To study the effect of small interfering RNA（siRNA）targeting Rac1b on the expression of angiogenesis-related molecules in gastric cancer AGS cells.Methods：siRNA targeting Rac1b（Rac1b siRNA）was synthesized and transfected into AGS cells by lipofectamineTM reagent.The effect of Rac1b siRNA on mRNA and protein expressions of Rac1b in AGS cells was examined by RT-PCR and Western blotting analysis.ELISA and Western blotting analysis were used to assess VEGF production in the supernatants of transfected AGS cells and the expressions of angiogenesis-related molecules such as P53,VHL and HIF-1α in AGS cells under hypoxia situation.Results：DNA sequencing analysis confirmed that the sequence of Rac1b siRNA was correct.Transfection of Rac1b siRNA in AGS cells resulted in a specific decrease in Rac1b mRNA and protein expressions,while failed to knock down the expression of its homologous molecular Rac1.The secretion of VEGF in the supernatant of AGS cells transfected with Rac1b siRNA was significantly inhibited,and this inhibition effect was more obvious under hypoxia condition.Meanwhile,Rac1b siRNA down-regulated HIF-1α protein expression and up-regulated the expressions of angiogenesis-related molecules such as P53 and VHL.Conclusion：Rac1b siRNA can inhibit the mRNA and protein expressions of Rac1b in gastric cancer AGS cells;it can also inhibit VEGF production through regulating the expression of angiogenesis-related molecules such as P53,VHL and HIF-1α under hypoxia condition.