中华医学遗传学杂志
中華醫學遺傳學雜誌
중화의학유전학잡지
Chinese Journal of Medical Genetics
2015年
4期
498-501
,共4页
王曦%李英慧%张红军%胡永胜%栾涛%陈芳杰
王晞%李英慧%張紅軍%鬍永勝%欒濤%陳芳傑
왕희%리영혜%장홍군%호영성%란도%진방걸
Smad泛素化调节因子1%核因子-κB%肝癌
Smad汎素化調節因子1%覈因子-κB%肝癌
Smad범소화조절인자1%핵인자-κB%간암
Smad ubiquitination regulatory factor 1%Nuclear factor-κB%Hepatic cancer
目的 鉴定Smad泛素化调节因子1(Smad ubiquitination regulatory factor 1,SMURF1)基因启动子区的一个核因子-κB(nuclear factor-κB,NF-κB)反应元件,并探讨后者对于SMURF1表达的调节作用.方法 构建系列截短的SMURF1基因启动子荧光素酶报告基因载体,转染肝癌Hep G2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-κB特异性小干扰RNA,检测SMURF1表达水平的变化.结果 SMURF1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SMURF1的表达水平.结论 NF-κB特异性结合于SMURF1启动子-411～-420区,对SMURF1的表达水平具有重要的调节作用.
目的 鑒定Smad汎素化調節因子1(Smad ubiquitination regulatory factor 1,SMURF1)基因啟動子區的一箇覈因子-κB(nuclear factor-κB,NF-κB)反應元件,併探討後者對于SMURF1錶達的調節作用.方法 構建繫列截短的SMURF1基因啟動子熒光素酶報告基因載體,轉染肝癌Hep G2細胞,檢測熒光素酶的活性,分析各段啟動子的活性.應用DNA結閤實驗和染色質免疫沉澱實驗鑒定SMURF1基因啟動子區的NF-κB反應元件;構建NF-κB位點突變的熒光素酶報告基因載體併檢測其活性;轉染NF-κB特異性小榦擾RNA,檢測SMURF1錶達水平的變化.結果 SMURF1基因的各段啟動子具有高轉錄活性,其中-519～-378區域為一箇正調控區;SMURF1啟動子-411～-420區域為NF-κB反應元件,NF-κB特異性結閤于該位點;該元件突變可使SMURF1啟動子活性明顯下降;轉染NF-κB特異性小榦擾RNA可下調SMURF1的錶達水平.結論 NF-κB特異性結閤于SMURF1啟動子-411～-420區,對SMURF1的錶達水平具有重要的調節作用.
목적 감정Smad범소화조절인자1(Smad ubiquitination regulatory factor 1,SMURF1)기인계동자구적일개핵인자-κB(nuclear factor-κB,NF-κB)반응원건,병탐토후자대우SMURF1표체적조절작용.방법 구건계렬절단적SMURF1기인계동자형광소매보고기인재체,전염간암Hep G2세포,검측형광소매적활성,분석각단계동자적활성.응용DNA결합실험화염색질면역침정실험감정SMURF1기인계동자구적NF-κB반응원건;구건NF-κB위점돌변적형광소매보고기인재체병검측기활성;전염NF-κB특이성소간우RNA,검측SMURF1표체수평적변화.결과 SMURF1기인적각단계동자구유고전록활성,기중-519～-378구역위일개정조공구;SMURF1계동자-411～-420구역위NF-κB반응원건,NF-κB특이성결합우해위점;해원건돌변가사SMURF1계동자활성명현하강;전염NF-κB특이성소간우RNA가하조SMURF1적표체수평.결론 NF-κB특이성결합우SMURF1계동자-411～-420구,대SMURF1적표체수평구유중요적조절작용.
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.
