RNA 干扰沉默 E637K 负显性突变基因效应的研究

摘要

Objective To investigate the effect of short interfering RNAs (siRNAs) on silencing of E637K-hERG mu-tant gene and its mechanisms. Methods Western blot was used to screen specific siRNAs targeting dominant-nega-tive mutant of E637K-hERG. Whole-cel patch-clamp technique was used to examine the changes of electrical current before and after siRNA transfection. Results siRNA significantly down-regulated the expression of protein bands by interference of E637K-hERG, but not by WT-hERG. Through inhibition of WT/E637K-hERG but not WT-hERG, siRNA in-creased activation current and tail current amplitudes, shifted the activation curve and steady-state inactivation curve to-ward positive potentials by 9.62mV and 17.41mV, respectively, and reduced the velocity of channel inactivation. Conclu-sion siRNA can effectively inhibit E637K-hERG protein expression and alter the properties of WT/E637K-hERG channel current.%　　目的探讨小干扰 RNAs(siRNAs)沉默 E673K-hERG 突变基因的效果及作用机制.方法 Western blot 筛选特异的 E637K-hERG 负显性突变基因靶向 siRNAs,全细胞膜片钳技术检测 siRNAs 干扰前后电流的变化.结果特异的 siRNAs 作用 E637K-hERG 后,蛋白质条带明显下调.而 WT-hERG 蛋白质条带无变化. siRNAs 干扰 WT /E637K-hERG 后,不仅使激活电流和尾电流的电流幅度都较干扰前明显增大,而且使激活电流和稳态失活电流的电向分别向正向移动了9.62mV 和17.41mV,减慢了通道电流失活速度.而 siRNAs 干扰 WT-hERG 前后电流幅度和特性无明显变化.结论 siRNAs 能有效抑制 E637K-hERG 的蛋白表达和改变 WT / E637K-hERG 的通道电流特性.