siRNA对HEK293细胞WT1表达的抑制作用及对K562细胞增生的影响  

Inhibitory effect of siRNA on WT1 expression in HEK293 cell and proliferation in K562 cell

作　　者：田彩霞[1] 王宏伟[1] 白波[1] 朱镭[1] 张丽[1] 李晓红[1] 

机构地区：[1]山西医科大学第二医院血液病研究所,太原030001

出　　处：《白血病．淋巴瘤》2008年第6期408-411,共4页Journal of Leukemia & Lymphoma

基　　金：山西省自然科学基金（20051102）

摘　　要：目的探讨以WT1基因为靶标治疗白血病的可行性，筛选有效抑制WT1基因表达的siRNA并观察其对K562细胞增生的影响。方法制备特异性WTi siRNA3条，转染HEK293细胞株，采用荧光定量RT—PCR方法检测WT1基因mRNA表达，Westernblotting法检测WT1蛋白表达；M1Tr法检测细胞增生的影响。结果不同位点的siRNA对WT1基因抑制作用不同。si—wt1-1对WT1mRNA抑制明显（P〈0．05），si—wt1-2、si—wt1-3对WT1mRNA无抑制（P〉0．05）。100nmol／L si—wt1—1能使WT1mRNA的表达在转染后24h和48h分别降低至对照组的（42．5±1．0）％（P〈0．05）、（25.3±1．5）％（P〈0．01），但72h恢复至对照组水平。siRNA作用无明显剂量依赖性，不同浓度si—wt1-1对WT1mRNA抑制作用的比较差异无统计学意义（P〉0．05）。Westernblotting法检测证实转染siRNA48、72、96h后，si—wt1-1对WT1蛋白抑制明显（P〈0．05），且96h抑制作用最强；si—wt1-2、si—wt1-3无明显抑制作用（P〉0．05）。si—wt1-1对K562细胞增生有抑制作用，si—wt1-1处理组与阴性对照组差异有统计学意义（P〈0．05）。结论siRNA可有效抑制HEK293细胞WT1基因的表达，且mRNA水平抑制效果优于蛋白水平。si—wt1-1可有效抑制K562细胞的增生。Objective To explore the feasibility of gene therapy targeting on WT1 gene in leukemia. siRNA inhibiting WT1 gene expression was effectively screened out and its affection on proliferation of K562 cell was observed. Methods Three siRNA for WT1 were designed and transfected into HEK293 cells. WT1 mRNA expression was detected by FQ-RT-PCR. WT1 protein expression was detected by Western blotting. The affection of cell proliferation was detected by MTT method. Results The inhibitory effects of siRNA designed different locations were different for wt1 gene. si-wt1-1 gene was the most significant （P 〈0.05）, but si-wt1-2 and si-wt1-3 had no inhibitory effect on WT1 mRNA expression（P 〉0.05）. WT1 mRNA expression was reduced to （42.5±1.0）% and （25.3±1.5）% of the controls at 24 h and 48 h transfected with 100 nmol/L si- wt1-1 respectively （P 〈0.05）, but restored to normal level at 72 h. There was no dose-dependent inhibitory effect for si-wt1-1 by the statistics analysis. The inhibitory effect of si-wt1-1 was obvious and the effect is best at 96h especially （P 〈0.05）. But si-wt1-2 and si-wt1-3 had no effect on WT1 protein expression by Western blolting ana]ysis（P 〉0.05）. si-wt1-1 had inhibitor）, effect for K562 cell proliferation,there was obvious difference between si-wt1-1 and negative control （P〈0.05）. Conclusion siRNA can effectively inhibit WT1 gene expression on HEK293 cells and the inhibitory effect on mRNA level is most significant on protein level. si-wt1-1 can effectively inhibit K562 cell proliferation.

关 键 词：RNA干扰 RNA 小分子干扰 基因 肾母细胞 

分 类 号：R733[医药卫生—肿瘤]