NF-κB对Smad泛素化调节因子1表达水平的调节作用

Regulation of Smad ubiquitination regulatory factor 1 expression by NF-κB

目的鉴定Smad泛素化调节因子1（Smad ubiquitination regulatory factor 1，SMURFl）基因启动子区的一个核因子-κB（nuclearfactor-κB，NF-κB）反应元件，并探讨后者对于SMURFl表达的调节作用。方法构建系列截短的SMURFl基因启动子荧光素酶报告基因载体，转染肝癌HepG2细胞，检测荧光素酶的活性，分析各段启动子的活性。应用DNA结合实验和染色质免疫沉淀实验鉴定SMURF1基因启动子区的NF-κB反应元件；构建NF-κB位点突变的荧光素酶报告基因载体并检测其活性；转染NF-xB特异性小干扰RNA，检测SMURF1表达水平的变化。结果SMURFl基因的各段启动子具有高转录活性，其中-519～-378区域为一个正调控区；SMURF1启动子-411--420区域为NF-κB反应元件，NF—κB特异性结合于该位点；该元件突变可使SMURF1启动子活性明显下降；转染NF-κB特异性小干扰RNA可下调SMURn的表达水平。结论NF-KB特异性结合于SMURF1启动子-411--420区，对SMURFl的表达水平具有重要的调节作用。

Objective To identify a nuclear factor-κB （NF-κB） responsive element within the Smad ubiquitination regulatory factor 1 （SMURF1）gene promoter, and to demonstrate its role in the regulation of SMURF1 expression. Methods A series of truncated luciferase reporter plasmids of the SMURF1 promoter were constructed and transfected into hepatic cancer Hep G2 cells. Luciferase assays were carried out to assess the activities of such promoters. DNA binding and chromatin immunoprecipitation （CHIP） assays were used to identify an NF-κB responsive element within the SMURF1 promoter. Lucifease plasmid with mutated NF-κB site was constructed and its activity was assessed. The expression of SMURF1 in Hep G2 cells was detected after transfection of NF-κB specific small interfering RNA （siRNA）. Results The SMURF1 promoter showed a high transcription activity, and the region of --519^- 378 was demonstrated to be a positive regulatory region. -- 411 ~ -- 420 of the SMURF1 promoter was an NF-κB responsive element, and NF-κB may specifically bind to this site. Mutation of this element may prominently decrease the activity of the promoter. Transfection of NF-κB siRNA evidently down-regulated SMURF1 expression. Conclusion NF-κB can specifically bind to the --411^--420 region of the SMURF1 promoter and plays an essential role in the expression of SMURF1.