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-α mRNA 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-α mRNA 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-α mRNA 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-α mRNA3'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-α mRNA稳定性无明显变化,蛋白表达明显上调；干扰HuR表达后,IκB-α mRNA稳定性无明显变化,蛋白表达明显下调.结论:(1) HuR可以与IκB-α mRNA 3'UTR特异性结合并参与调节IκB-α mRNA 3'UTR的功能；(2) HuR对IκB-α mRNA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-κB inhibitor α(IκB-α) by RNA-binding protein HuR at post-transcriptional level. METHODS: The vector containing IκB-α mRNA 3'UTR reporter gene was constructed and transfected into NIH 3T3 cells. 3'UTR of IκB-α mRNA was labeled with biotin in vitro and RNA pull-down assay was performed. Under the conditions of overexpression or silencing of HuR, the mRNA stability of IκB-α was detected by qPCR and the protein level of IκB-α was examined by Western blotting. RESULTS: The eukaryotic expression vector of lucifer-ase reporter gene for 3'UTR of IκB-α mRNA was correctly constructed. pcDNA3-HA-HuR plasmid and IκB-α mRNA 3'UTR lucifERαse reporter gene were cotransfected into 3T3 cells. The lucifERαse activity in the transfected cells was increased compared with control group. The results of RNA pull-down assay with biotin-labeled IkB-o: mRNA 3'UTR and Western blotting confirmed the binding protein of HuR. After overexpression of HuR for 24 h, no significant influence on IκB-α mRNA stability but significant up-regulation of IκB-α protein expression was observed. Knock-down of HuR expres-rnsion with siRNA did not change the stability of IκB-α mRNA but down-regulated the protein expression of IκB-α. CONCLUSION: HuR is able to bind to 3'UTR of IκB-α mRNA specifically and regulates IκB-α mRNA 3'UTR function. HuR up-regulates the protein expression of IκB-α without influencing IκB-α mRNA stability.
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