葛根素对子宫内膜RL95-2细胞P450芳香化酶表达的调控作用(英文)

Regulation of aromatase P450 expression by puerarin in endometrial cell line RL95-2

目的:探讨葛根素对子宫内膜RL95-2细胞P450芳香化酶(aromatase P450,P450arom)表达的影响及其对RL95-2细胞P450arom基因表达的调控作用。方法:将梯度稀释的葛根素在不同时间点加入RL95-2细胞中,分别采用实时聚合酶链反应(real-time polymerase chain reaction,RT-PCR)检测药物对RL95-2细胞P450arom mRNA表达水平的影响。以人类基因组DNA为模板扩增人P450arom组织特异性启动子2(PⅡ)转录起始位点上游序列。PCR产物定向克隆到报告基因载体pGL3-Basic中,构建人P450aromPⅡ系列报告基因质粒,并经限制性内切酶消化鉴定和基因测序证实。瞬时转染系列重组质粒至RL95-2细胞,加入葛根素刺激,分析相关转录因子,利用RT-PCR及Western blotting方法检测相关转录因子在葛根素作用下mRNA及蛋白表达的变化。结果:与溶剂对照组比较,低浓度葛根素会抑制P450arom mRNA表达(P<0.01),抑制作用在6、12 h较显著(P<0.01)。经酶切鉴定、基因测序、转录因子分析及启动子活性测定,成功构建了人P450aromPⅡ系列重组质粒(-1 017,-763,-543,-234^+8 bp)。瞬时转染系列质粒至细胞中,于12 h后加入葛根素(10-7mol/L)刺激,又12 h后检测荧光蛋白水平。结果表明,-763^-543 bp间的活性被抑制(P<0.05),通过转录因子分析找到了-410/-401 bp的AP-1(c-jun/c-fos)。利用RT-PCR、Western blotting及荧光素酶活性检测发现葛根素(10-7mol/L)对AP-1(c-jun/c-fos)的抑制作用平行于对P450arom的作用。结论:低浓度葛根素对子宫内膜RL95-2细胞P450芳香化酶基因表达的抑制作用很可能是受上游调控转录因子AP-1(c-jun/c-fos)的影响。

Objective： To study the effects of puerarin on the aromatase P450（P450arom） mRNA expression and the effects of low-dose puerarin on transcription factors of the P450arom gene（PⅡ） 5＇-flanking region.Methods： The effects of puerarin on the P450arom mRNA expression were determined by real-time polymerase chain reaction（RT-PCR）.The 5＇-flanking region was amplified by PCR using human genomic cDNA as a template.By means of the restriction sites and sequence confirmation,the PCR product was cloned into reporter vector.Series of sequential deletion reporter constructs were transiently transfected into RL95-2 cells which were treated with or without puerarin.Luciferase activity was measured by Dual-Luciferase Reporter Assay System and Luminoskan Ascent luminometer.Furthermore,by using web-based search program,the most possible cis-acting elements and transcription factors were evaluated.Results： The data demonstrated that low-dose puerarin treatment could decrease P450arom expression at mRNA level compared to dimethyl sulphoxide（DMSO） treatment（P〈0.01）,and puerarin（10^-7 mol/L） had a time-course effect on P450arom mRNA expression,which reached the bottom at 12 h（P〈0.01）.Cells transfected with the-763/＋8 bp constructs showed decrease in relative luciferase activity after puerarin（10^-7 mol/L） treatment compared to DMSO treatment（P〈0.05）,indicating an essential regulatory site between-763 bp and-543 bp responsible for the transcription suppression by puerarin.Furthermore,the most possible transcription factors,which turned out to be AP-1（c-jun/c-fos） at-410/-401 bp were also evaluated.The activity of exogenous AP-1 was reduced after 12 hours of puerarin treatment（P〈0.05）.The inhibition of c-jun mRNA also showed a time-course effect,which bottomed out at 12 h in parallel with that of P450arom（P〈0.01）.The protein level of c-jun was also down-regulated by puerarin（10^-7mol/L） treatment at 12 h.Conclusion： The suppression of P450arom expression and activity may be associated with the down-regulation of transcription factor AP-1/c-jun.This partially explains the mechanisms whereby puerarin treats endometriosis.