RNA干扰诱导型一氧化氮合酶基因表达对人胆管癌细胞生长的影响

目的:探讨RNA干扰诱导型一氧化氮合酶(iNOS)基因的表达对人胆管癌细胞生长的影响。方法:针对iNOS设计并合成3种iNOS-siRNA序列(siRNA1、siRNA2、siRNA3)及阴性对照siRNA序列后,分别转染人胆管癌细胞QBC939细胞,用荧光显微镜下观察转染效率,通过iNOS mRNA与蛋白的变化分析干扰效果,选择干扰效果最为明显的iNOS-siRNA序列,观察其干扰后QBC939细胞增殖、细胞周期及细胞凋亡的变化。实验以无处理的QBC939细胞为空白对照。结果:合成的3种iNOS-siRNA序列均可有效转染人胆管癌QBC939细胞,且转染后均能明显降低QBC939细胞中iNOS mRNA与蛋白的表达(均P0.05),其中siRNA2对iNOS的抑制作用最为明显,阴性对照siRNA序列对QBC939细胞中iNOS mRNA与蛋白的表达无明显影响(均P0.05)。转染siRNA2后,QBC939细胞增值明显降低、出现明显的G0/G1期阻滞、凋亡率明显增加(均P0.05);转染阴性对照siRNA序列的QBC939细胞无上述改变(均P0.05)。结论:RNA干扰能有效降低iNOS基因在胆管癌细胞中的表达,从而抑制胆管癌细胞的增殖,并促进其凋亡。

Effect of RNA interference of inducible nitric oxide synthase gene expression on growth of human cholangiocarcinoma cells

Objective: To investigate the effect of RNA-mediated interference of inducible nitric oxide synthase (iNOS) gene expression on growth of human cholangiocarcinoma cells.Methods: Three iNOS-siRNA sequences targeting iNOS gene (siRNA1, siRNA2 and siRNA3) and a negative control siRNA sequence were designed and synthesized, and were respectively transfected into human cholangiocarcinoma QBC939 cells, and then, the transfection efficiencies were determined by fluorescence microscopic observation, and the interference effects were examined through changes in iNOS mRNA and protein expressions. The iNOS-siRNA sequence with most evident interference efficacy was selected for use to observe the changes in proliferation, cell cycle and apoptosis in QBC939 cells after its transfection. The untreated QBC939 cells served as blank control.Results: All the three synthesized iNOS-siRNA sequences were effectively transfected into QBC939 cells, and iNOS mRNA and protein expression levels were all significantly reduced in QBC939 cells after their transfection (all P<0.05), in which, siRNA2 showed the most evident inhibitory effect on iNOS. The negative control siRNA sequence exerted no significant effect on iNOS mRNA and protein expressions in QBC939 cells (both P>0.05). In QBC939 cells after siRNA2 transfection, the proliferation was significantly decreased, with significant G0/G1 phase arrest and increased apoptosis rate (all P<0.05), while no significant changes were noted in above indexes in QBC939 cells after transfection of negative control siRNA sequence (all P>0.05). Conclusion: RNA interference can effectively decrease iNOS gene expression in cholangiocarcinoma cells, which thereby inhibit the proliferation and promote apoptosis of cholangiocarcinoma cells.