RNA结合蛋白HuR对NF-κB抑制因子α转录后调控机制的初步研究  

Regulation of IκB-α by RNA-binding protein HuR at post-transcriptional level

作　　者：于文燕[1] 马小娟[1] 孙宏卫[2] 买买提祖农·买苏尔[1] 马瑞斌[3] 柯颖[4] 刘玉新[5] 马琪[6] 

机构地区：[1]新疆医科大学病理生理学教研室 [2]新疆医科大学南方医科大学病理生理学教研室,广东广州510515 [3]武警新疆总队医院普外科,新疆乌鲁木齐830000 [4]武警新疆总队医院神经外科,新疆乌鲁木齐830000 [5]宁波大学医学院组织学教研室,浙江宁波315000 [6]新疆医科大学机能中心,新疆乌鲁木齐830000

出　　处：《中国病理生理杂志》2013年第4期688-694,共7页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81171476);浙江省杰出青年科学基金资助项目(No.R2101166);宁波市创新团队项目(No.2011B82014)

摘　　要：目的:探讨RNA结合蛋白HuR对NF-κB抑制因子α(IκB-α)转录后调控机制。方法:构建IκB-αmRNA 3'UTR报告基因真核表达质粒,并转染3T3细胞;体外生物素标记IκB-αmRNA 3'UTR并进行RNA pull-down;过表达及干扰HuR后,qPCR检测其对IκB-αmRNA稳定性的影响,Western bltting检测其对IκB-α蛋白表达的影响。结果:(1)IκB-αmRNA 3'UTR报告基因真核表达质粒构建成功;(2)报告基因检测:共转染pcDNA3-HA-HuR质粒及IκB-αmRNA 3'UTR报告基因质粒后与对照组相比其数值明显增高;(3)用生物素标记的IκB-αmRNA3'UTR探针进行RNA pull-down,可以检测到特异性结合的HuR蛋白;(4)过表达HuR,IκB-αmRNA稳定性无明显变化,蛋白表达明显上调;干扰HuR表达后,IκB-αmRNA稳定性无明显变化,蛋白表达明显下调。结论:(1)HuR可以与IκB-αmRNA 3'UTR特异性结合并参与调节IκB-αmRNA 3'UTR的功能;(2)HuR对IκB-αmRNA稳定性无明显影响,其主要调控IκB-α蛋白表达,使其表达上调。AIM： To investigate the regulation of NF-KB inhibitor α（IKB-α4） by RNA-binding protein HuR at post-transcriptional level. METHODS： The vector containing IKB-α mRNA 3＇UTR reporter gene was constructed and transfected into NIH 3T3 cells. 3＇UTR of IKB-ot mRNA was labeled with biotin in vitro and RNA pull-down assay was per- formed. Under the conditions of overexpression or silencing of HuR, the mRNA stability of IKB-α was detected by qPCR and the protein level of IKB-α was examined by Western blotting. RESULTS ： The eukaryotie expression vector of lucifer- ase reporter gene for 3＇UTR of IKB-ct mRNA was correctly constructed, pcDNA3-HA-HuR plasmid and IKB-ct mRNA 3＇UTR luciferase reporter gene were cotransfected into 3T3 ceils. The luciferase activity in the transfected cells was in- creased compared with control group. The results of RNA pull-down assay with biotin-labeled IKB-ot mRNA 3＇UTR and Western blotting confirmed the binding protein of HuR. After overexpression of HuR for 24 h, no significant influence on IKB-ot mRNA stability but significant up-regulation of IKB-ct protein expression was observed. Knock-down of HuR expres-sion with siRNA did not change the stability of IKB-ot mRNA but down-regulated the protein expression of IKB-α. CON- CLUSION： HuR is able to bind to 3＇UTR of IKB-α mRNA specifically and regulates IKB-α mRNA 3＇UTR function. HuR up-regulates the protein expression of IKB-α without influencing IKB-α mRNA stability.

关 键 词：HuR蛋白 NF-κB抑制因子α 转录后调控 

分 类 号：R363.2[医药卫生—病理学]