siRNAs表达载体沉默RAB5A基因的位置效应研究  

Positional effect of short interfering RNAs expressing vector targeting human RAB5A

作　　者：陈香梅[1] 李蒙[1] 刘珊珊[1] 李钰[1] 

机构地区：[1]哈尔滨医科大学医学遗传学研究室,150081

出　　处：《中国地方病学杂志》2006年第6期699-702,共4页Chinese Jouranl of Endemiology

基　　金：国家自然科学基金资助项目(39970396);黑龙江省攻关项目留学回国基金资助(LC04C02);黑龙江省教育厅海外学人科研(合作)项目资助(1054HZ013)

摘　　要：目的研究小干扰RNA(siRNAs)表达载体沉默RAB5A基因的位置效应,寻找沉默RAB5A基因的最佳作用靶点。方法利用MFold软件分析RAB5A mRNA的二级结构,构建针对RAB5A mRNA不同位点的siRNAs表达载体,转染Anip973细胞后,通过RT-PCR和Western-blot方法评价siRNAs对RAB5A基因表达的抑制作用。结果针对RAB5A mRNA编码区内最可及位点设计的siRNAs分别抑制72％和85％的RAB5A基因表达,而针对最不可及位点设计的siRNAs则完全没有活性;非编码区内也存在沉默RAB5A基因的siRNAs有效作用靶点;siRNAs分子与RAB5A mRNA靶序列之间引入2个错配碱基时,siRNAs的抑制活性完全丧失。结论MFold软件分析的RAB5A mRNA二级结构可以作为分析和寻找可及位点的依据,siRNAs的作用效率与RAB5A mRNA靶位点的可及性密切相关。Objective To study the structural effect of short interfering RNAs （siRNAs） expressing vector targeting human RABSA, find the most effective target site of RABSA mRNA. Methods The secondary structure of RABSA mRNA was predicted using the MFold software. Several siRNAs expressing vectors against different sites of RABSA mRNA were synthesized and transfected into Anip973. Inhibition of RABSA gene expression was detected by RT-PCR and Western-blot. R^uits siRNAs targeting the most accessible sites inhibited 72% and 85% of RABSA expression, while those targeting the least accessible sites were completely inactive. Effective targeting site of siRNAs was also found in the untranslating region of RABSA mRNA. Two mismatched base pairs between siRNA and target sequence resulted in a significant reduction of siRNA activity. Conclusions The secondary structure of RAB5A mRNA analyzed by the software MFold can be used to find the accessible sites, and the efficiency of siRNAs depends on the accessibility of target sites of RAB5A mRNA.

关 键 词：RAB5A基因 小干扰RNA 位置效应 可及位点 

分 类 号：R734.2[医药卫生—肿瘤]