中文摘要：

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。

英文摘要：

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.