| Endometriosis is a common estrogen-dependent disease characterized by the presence of functional endometrium outside the uterus.Aromatase P450(P450arom) is a key enzyme for biosynthesis of estrogen which is an essential hormone for the establishment and growth of endometriosis.Aromatase inhibitors have successfully been used to treat endometriosis and uterine leiomyomata.There was also evidence that phytoestrogens and their low dose combinations inhibited mRNA expression and activity of aromatase in human granulosa-luteal cells.Puerarin is a major isoflavonoid compound isolated from Pueraria lobata,a Chinese medicine known as Gegen.Based on the above reasons,we hypothesized that puerarin may have effects on estrogen or aromatase inhibition as other isoflavonoids.Therefore,this study was carried out to elucidate the inhibitory potential and mechanisms of puerarin on EM cells with P450arom overexpression.Objectives:①To study the effect of Puerauin on the P450arom expression and activity.②The effect of low dose puerarin on transcription factors of the P450arom gene(PII) 5'-flanking region.③siRNA downregulation associated transcription factors attenuated P450arom expression and activity.Methods:①The effects of puerauin on the P450arom mRNA and protein expression were determined by real-time PCR and western blotting assays.To further evaluate whether puerarin affects P450arom activity,estradiol assasys were used.②The 5'-flanking region was amplified by PCR using human genomic cDNA as a template. Using the restriction sites and sequence confirmation,the PCR product was cloned into reporter vector.Putative transcription factor binding sites within the full-length P450arom PII were identified using the web-based search program.Series of sequential deletion reporter constructs were transiently transfected into RL95-2 cells which were treated with or without puerarin.Luciferase activity was measured using the Dual-Luciferase Reporter Assay System and a Luminoskan Ascent luminometer. Furthermore,we used web-based search program to evaluate the most possible cis-acting elements and transcription factors.③To further confirm if the associated transcription factors are responsible for the P450arom regulation,we next knocked down them expression using siRNA,and then determined its effect on P450arom.Results:①Our data demonstrated puerarin may exert two-way properties.Low dose puerarin treatment decrease P450arom expression at mRNA levels compared to DMSO treatment(P＜0.01) and puerarin(10-7mol/L) had a time-course effect on P450arom mRNA expression,which was reached the bottomat 12h(P＜0.01)., Meanwhile,P450arom protein levels were obviously decreased(＞50%) by 12hrs puerarin(10-7mol/L) treatment.Moreover,puerarin diminished E2 levels though not as remarkably as positive control lestrozole(P＜0.05).But,the increase of P450arom expression at both mRNA and protein levels by high dose puerarin treatment compared to DMSO treatment(P＜0.01).Also puerarin(10-3mol/L) strengthen P450arom activity at 48h compared to DMSO treatment(P＜0.05).②After sequence confirmation,a series of sequential deletion reporter constructs(-1017,-763,-543 and -234 to +8bp) were cloned into the luciferase-containing vector pGL3-basic. Cells transfected with the -763/+8bp constructs with puerarin(10-7mol/L) showed decrease in relative luciferase activity compared to DMSO treatment(P＜0.05), suggesting an essential regulatory site existed between -763bp and -543bp responsible for the transcription suppression by puerarin.Furthermore,we evaluate the most possible transcription factors,which turned out to be AP-1(c-jun/c-fos) at -410/-401bp.The activity of exogenous AP-1 was reduced after 12hrs puerarin treatment(P＜0.05).The inhibition of c-jun mRNA also showed a time-course effect, which bottomed out at 12h in parallel with that of P450arom(P＜0.01).The protein level of c-jun was also downregulated by puerarin(10-7mol/L) treatment at 12h.③The results shown mRNA levels of c-jun and P450arom were both significantly reduced by c-jun siRNA(P＜0.01) compared with scrambled siRNA treatment.Besides,the protein level of P450arom also showed a 50%inhibition confirmed by western blot assay.In addition to the regulation of P450 expression our data proved that knocking-down c-jun leaded to aromatase deficiency in converting testosterone to estradiol.Conclusions:①The effect of prauin on the P450arom expression and activity may exert different properties.Low dose puerarin decrease P450arom expression and activity;otherwise high dose puerarin may increase P450arom expression and activity.②The P450arom gene(PII) region and deletion constructs were successfully constructed.The structures for all vectors were confirmed by digestion with restriction endonucleases,followed by DNA sequence analysis.The essential transcription factors may exist between -410/-401bp may associate with c-jun and AP-1(c-jun/c-fos).When puerarin was used,c-jun was down regulated and so did P450arom.③We demonstrated that in untreated RL95-2 cells,when c-jun was knocked down,the expression of P450arom exhibited＞50%decrease at both mRNA and protein level,which means c-jun is essential for P450arom overexpression.
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