干扰HERG钾通道表达可抑制成神经细胞瘤细胞增殖

目的：采用RNA干扰（RNA interference，RNAi）技术抑制HERG基因的表达，探讨HERG基因编码钾通道对人成神经细胞瘤细胞（SH-SY5Y）存活及增殖的影响。方法：构建发卡状RNA干扰质粒（shRNA-HERG）转染SH-SY5Y细胞，再分别使用半定量RT-PCR和Western印迹方法观察shRNA-HERG对SH-SY5Y细胞中HERG基因mRNA及其编码蛋白表达的干扰效果。利用SH-SY5Y细胞的生长曲线和集落形成实验，观察转染shRNA-HERG质粒对SH-SY5Y细胞存活及增殖能力的影响。结果：转染shRNA-HERG干扰质粒后，SH—SY5Y细胞中HERG基因的mRNA水平及其编码的钾通道蛋白表达水平显著下降。与对照组相比，shRNA—HERG使SH-SY5Y细胞生长曲线明显下压，细胞倍增时间延长136．8％。集落形成实验结果显示，无论接种细胞数为50，100或200，shRNA-HERG组克隆形成率均显著下降，与shRNA-control组和未干扰组相比，差异有统计学意义（P〈0．01）。结论：所构建的shRNA-HERG表达质粒能有效抑制HERG基因编码钾通道蛋白的表达，明显抑制SH-SY5Y细胞的生长和增殖。

Interfering expression of HERG potassium channels depresses existence and proliferation of neuroblastoma cells

Objective: To explore the relationship between HERG potassium channels and existence and proliferation of neuroblastoma(SH-SY5Y) cells using RNA inference technique.Methods: Expression vector of HERG gene-targeting small hairpin interfering RNA(shRNA) was constructed(shRNA-HERG).The interfering effect of shRNA-HERG on the expression of HERG mRNA and protein was determined by half-quantitative RT-PCR and Western blot. The growth and proliferation of SH-SY5Y cells were examined by cell growth curve and colony forming experiment.Results: After SH-SY5Y cells were transfected with shRNA-HERG,the expression of HERG gene and protein was suppressed remarkably.The growth curve of SH-SY5Y cells was depressed and the growth doubling time was prolonged by 136.8%.The ability of colony forming was decreased compared with shRNA-control and untreated groups,whatever 50,100 or 200 cells were inoculated.Conclusion: The shRNA-HERG can depress the expression of HERG gene and proteins,and inhibit the growth and proliferation of SH-SY5Y cells.