siRNA靶向下调nNav1.5影响胶质瘤细胞增殖与凋亡研究

siRNA-targeted down-regulating n Nav1.5 expression affects the proliferation and apoptosis of glioma cells

目的研究下调电压-门控钠离子通道(VGSCs)幼稚型钠离子通道1.5(n Nav1.5)的表达对胶质瘤U251细胞增殖和凋亡的影响。方法用免疫荧光技术检测n Nav1.5在U251细胞的表达;设计合成n Nav1.5的靶向小干扰核糖核酸(siRNA),脂质体瞬时转染至U251细胞,用实时定量逆转录酶-聚合酶链反应(Real-time RT-PCR)和蛋白免疫印迹法(Western blot)分别检测n Nav1.5的mRNA和蛋白水平变化,并判断siRNA干扰效果;用四唑盐比色法(MTT)和流式细胞仪检测siRNA对细胞增殖和凋亡的影响。结果 n Nav1.5主要在U251细胞核中表达;siRNA可显著下调n Nav1.5的mRNA和蛋白表达水平,并抑制细胞增殖、促进细胞凋亡。结论 n Nav1.5在胶质瘤的发生发展过程中起着促进作用,可能成为胶质瘤诊断和治疗的一个新靶点。

Objective The effect of down-regulating voltage-gated sodium channel（VGSC） alpha subunit neonatal Nav1. 5（ n Nav1. 5） on proliferation and apoptosis of glioma U251 cells is studied. Methods Immunofluorescence was used to localize the n Nav1. 5 protein in U251 cells. The small interfering ribose nucleic acid（ siRNA） targeting human n Nav1. 5 gene was designed and transfected transiently into U251 cells by lipofectamine. Real-time reverse transcription-polymerase chain reaction（ Real-time RT-PCR） and Western blot method were used to detect the changes of expressions of n Nav1. 5 mRNA and protein as well as to evaluate the efficacy of RNA interference. Mosmann tetrazolium test（ MTT） assay and flow cytometric detection were used to detect cell proliferation and apoptosis,respectively. Results The n Nav1. 5 protein mainly localized in nucleus of U251 cells. The expressions of n Nav1. 5 mRNA and protein decreased obviously in U251 cells after the siRNA transfection. And the proliferation in vitro was remarkably decreased and the apoptosis rate was significantly increased in U251 cells after the siRNA transfection. Conclusion n Nav1. 5plays a conductive role in the development of glioma,which might become a new molecular marker in tumor diagnosis and treatment.