Role Of Estrogen Responsive Ring Finger Protein On The Angiogenesis Of Endometrial Carcinoma

Objective:To investigate the interaction of Efp expression on different endometrial carcinoma cells and the role of estrogen and selective estrogen receptor modulators.Methods:we performed growth studies of 17β-estradiol(E2)and SERMs(4OHT and raloxifene)on estrogen receptor positive Ishikawa and negative HEC-1B cells. The effects of E2 and SERMs on the induction of VEGF and estrogen-responsive finger protein(Efp)were measured by QRT-PCR and ELISA or western blot.The siRNA method was used to investigate the action of Efp on Ishikawa cell growth and the induction of VEGF by E2 and SERMs.Results:E2 and 4OHT induced Ishikawa cell proliferation,the expression of Efp mRNA and protein increased 2 times with treatment by E2(10^-8M)for 24h,and that of VEGF increased 2.85 times(P＜0.001)and 2.10 times(P＜0.05)compared with control. Meanwhile,the expression of Efp mRNA and protein increased 1.5 times with treatment by 4OHT(10^-7TM)for 24h,and that of VEGF increased 1.94 times(P＜0.05) and 1.90 times(P＜0.05)compared with control.while raloxifene did not.The effects of estradiol were partly or almost completely inhibited by tamoxifen and raloxifene. The shRNA could suppress the expression of Efp mRNA and protein effectively,the expression of Efp mRNA decreased about 80%at 48h after transfection.When the Efp expression was suppressed by siRNA,the cell growth was decreased with estradiol treatment,and the induction of VEGF by estradiol and tamoxifen were decreased.Estradiol could also induce HEC-1B cell growth and expression of VEGF on mRNA and protein levels,the expression of VEGF mRNA and protein increased 2.35 times and 2.46 times(P＜0.05)with treatment by E2(10^-8M)for 24h compared with control,however,tamoxifen and raloxifene could not.Conclusion:These results demonstrate that E2 and 4OHT may regulate the growth of endometrial carcinoma Ishikawa cells by stimulating VEGF production through Efp,and the effects of E2 could be amplified by Efp.This study suggest that 4OHT acts as an agonist-antagonist,and raloxifene acts as an antagonist of estrogen in Ishikawa cells in vitro.E2 could induce the HEC-1B cell growth and expression of VEGF through other pathways. Objective:To investigate the effects of estradiol and SERMs(4OHT and raloxifene)on regulation of basic fibroblast growth factor in endometrial carcinoma cells in vitro.Methods:The effects of estradiol,4OHT and raloxifene on the induction of bFGFmRNA and protein were measured by QRT-PCR and ELISA methods.Three shRNA were synthesized targeting the Efp gene and transfected into the endometrial carcinoma Ishikawa cells using Lipofectamine 2000.The induction of bFGF by estradiol and SERMs were measured by the same methods after transfection in Ishikawa cells.Results:Estradiol induced expression of bFGF on mRNA and protein levels in endometrial cancer cells.Estradiol increased the expression of bFGFmRNA 2.52±0.16 times,and the secretion of the bFGF protein was(24.2±1.4)pg/ml,which were all higher than that of the control in HEC-1B cells(P＜0.05).Meanwhile, estradiol could increase the expression of bFGFmRNA 2.56±0.17 and 2.14±0.06 times,and the secretion of the bFGF protein was(26.24±2.2)pg/ml and(23.7±1.2) pg/ml in Ishikawa cells before and after Efp siRNA transfection,which were all higher than those of controls(P＜0.05).Raloxifene and 4OHT could not regulate the expresson of bFGF,and the regulation of bFGF by estradiol was inhibited by 4OHT and raloxifene. Conclusion:These results demonstrate that the regulation of bFGF by estradiol were not Efp depended. Objective:To investigate the expression of estrogen-responsive ring finger protein(Efp),vascular endothelial growth factor(VEGF)and basic fibroblast growth factor(bFGF)on endometrial cancer and the relation to the clinic-pathologic manifestitions.Methods:The levels of protein and messenger RNA(mRNA)expression of VEGF,bFGF and Efp in human endometrial tissue(normal endometriun[NE,n=20]; simple hyperplasia[SH,n =10];complex hyperplasia[CH,n =10];atypical hyperplasia[AH,n =10]and endometrioid adenocarcinoma[EC,n=35])were measured using quatitative real-time polymerase chain reaction,enzyme linked immunosorbent assay(ELISA)and western blot.Results:The expression of Efp mRNA was significantly decreased in the groups of EC in comparison with the groups of NE(P＜0.05).Expression of VEGF mRNA in the groups of EC and AH were(3.20±0.45)and(2.51±0.37),that was significantly increased in comparison with the groups ofNE(P＜0.05).The level of bFGFmRNA expression in groups of EC and AH were(6.43±0.73)and(3.46±0.62),that was higher than that ofNE groups((P＜0.01 and P＜0.05),and the level of EC was higher than that of SH groups(P＜0.05).The highest expression was observed in EC.There was positive relevance of bFGF expression and histologic grade,and negative relevance of Efp expression and histologic grade.No relevance was found with regard to FIGO stage.The levels of VEGF,bFGF protein in groups of EC were (35.25±3.13)ng/ml and(45.26±3.79)ng/ml,that was higher that that of NE and SH (P＜0.05).The expression of Efp protein of EC group was(28.60±2.95),which decreased compared with that of NE and SH(P＜0.05).Conclusion:The high expression of VEGF and bFGF indicate that both of them contribute in the angiogenesis of endometrial cancer.VEGF may play a major role in the early stage of endometrial cancer,and in the advanced stage and poorly differentiated endometrial cancer,bFGF may play the major role.The regulation of bFGF and Efp expression during endometrial carcinogenesis suggests their potential utility as a prognostic biomarker.