缺氧环境下外源性雌激素对子宫内膜癌细胞上皮间充质转化的影响及其机制

Effects of exogenous estrogen on epithelial mesenchymal transformation of endometrial cancer cells under hypoxia and its mechanism

目的探讨缺氧环境下外源性雌激素对子宫内膜癌细胞上皮间充质转化(EMT)的影响及其机制。方法选择雌激素受体阳性的子宫内膜癌细胞Ishikawa,体外传代培养。取传3代、对数生长期、生长状态良好的Ishikawa细胞进行后续实验。取部分Ishikawa细胞,于0.5%O_(2)、5%CO_(2)、94.5%N2环境中缺氧预处理2 h,随机分为siRNA-缺氧诱导因子1α(HIF-1α)组与siRNA-NC组、HIF-1α过表达组与Control组,siRNA-HIF-1α组、siRNA-NC组、HIF-1α过表达组分别转染siRNA-HIF-1α、siRNA-NC、HA-HIF-1α-pcDNA3,Control组不予转染;另取部分Ishikawa细胞,于0.5%O_(2)、5%CO_(2)、94.5%N2环境中缺氧预处理2 h后加入0.01μmol/Lβ-雌二醇,随机分为Estrogen组、Estrogen+siRNA-NC组、Estrogen+siRNA-HIF-1α组以及Control组、Estrogen+HIF-1α过表达组,Estrogen+siRNA-NC组、Estrogen+siRNA-HIF-1α组、Estrogen+HIF-1α过表达组分别转染siRNA-NC、siRNA-HIF-1α、HA-HIF-1α-pcDNA3。转染24 h,收集各组细胞,分别采用实时荧光定量PCR法、Western blotting法检测HIF-1α及EMT相关基因(E-cadherin、N-cadherin、β-catenin、TGF-β_(1))mRNA和蛋白表达。结果与siRNA-NC组比较,siRNA-HIF-1α组HIF-1α、N-cadherin、β-catenin、TGF-β_(1)mRNA和蛋白相对表达量均降低,E-cadherin mRNA和蛋白相对表达量均升高(P均<0.05);与Control组比较,HIF-1α过表达组HIF-1α、N-cadherin、β-catenin、TGF-β_(1)mRNA和蛋白相对表达量均升高,E-cadherin mRNA和蛋白相对表达量均降低(P均<0.05)。Estrogen+siRNA-HIF-1α组HIF-1α、N-cadherin、β-catenin、TGF-β_(1)mRNA和蛋白相对表达量均低于Estrogen+siRNA-NC组和Estrogen组,E-cadherin mRNA和蛋白相对表达量均高于Estrogen+siRNA-NC组和Estrogen组(P均<0.05);Estrogen+HIF-1α过表达组HIF-1α、N-cadherin、β-catenin、TGF-β_(1)mRNA和蛋白相对表达量均高于Estrogen组和Control组,E-cadherin mRNA和蛋白相对表达量均低于Estrogen组和Control组(P均<0.05)。结论缺氧环境下外源性雌激素可能通过激活HIF-1α来促进子宫内膜癌细胞EMT。

Objective To investigate the effect of exogenous estrogen on epithelial mesenchymal transformation(EMT)of human endometrial cancer cells under hypoxia and its mechanism.Methods Endometrial cancer cells Ishikawa with positive estrogen receptor were selected and cultured in vitro.Ishikawa cells in the third passage,which were in loga⁃rithmic growth phase and had good growth status,were taken for the follow-up experiments.Some Ishikawa cells were pre⁃treated with hypoxia in the environment of 0.5%O_(2),5%CO_(2)and 94.5%N2 for 2 h,and were randomly divided into the siRNA-HIF-1αgroup,siRNA-NC group,HIF-1αoverexpression group and Control group.Cells in the siRNA-HIF-1αgroup,siRNA-NC group,and HIF-1αoverexpression group were transfected with siRNA-HIF-1α,siRNA-NC,and HA-HIF-1α-pcDNA3,respectively,while cells in the Control group were not transfected.Other Ishikawa cells were pre⁃treated with 0.5%O_(2),5%CO_(2)and 94.5%N2 for 2 h and then were added with 0.01μmol/Lβ-estradiol.They were ran⁃domly divided into the Estrogen group,Estrogen+siRNA-NC group,Estrogen+siRNA-HIF-1αgroup,Control group,and Estrogen+HIF-1αoverexpression group.Cells in the Estrogen+siRNA-NC group,the Estrogen+siRNA-HIF-1αgroup,and the Estrogen+HIF-1αoverexpression group were transfected with siRNA-NC,siRNA-HIF-1α,and HA-HIF-1α-pcDNA3,respectively.The cells were collected at 24 h after transfection.Real-time fluorescent quantitative PCR and Western blotting were used to detect the mRNA and protein expression levels of HIF-1αand EMT-related genes(E-cadherin,N-cadherin,β-catenin,and TGF-β_(1)),respectively.Results Compared with the siRNA-NC group,the mRNA and pro⁃tein relative expression levels of HIF-1α,N-cadherin,β-catenin,and TGF-β_(1)in the siRNA-HIF-1αgroup significantly decreased,while the mRNA and protein relative expression levels of E-cadherin significantly increased(all P<0.05).Compared with the Control group,the mRNA and protein relative expression levels of HIF-1α,N-cadherin,β-catenin and TGF-β_(1)in the HIF-1αoverexpression group significantly increased,while the mRNA and protein relative expression levels of E-cadherin significantly decreased(all P<0.05).The relative mRNA and protein expression levels of HIF-1α,N-cad⁃herin,β-catenin and TGF-β_(1)in the Estrogen+siRNA-HIF-1αgroup were significantly lower than those in the Estrogen+siRNA-NC group and Estrogen group;the mRNA and protein relative expression levels of E-cadherin were significantly higher than those of the Estrogen+siRNA-NC group and Estrogen group(all P<0.05).The relative mRNA and protein expression levels of HIF-1α,N-cadherin,β-catenin and TGF-β_(1)in the Estrogen+HIF-1αoverexpression group were sig⁃nificantly higher than those in the Estrogen group and Control group;the mRNA and protein relative expression levels of E-cadherin were significantly lower than those of the Estrogen group and Control group(all P<0.05).Conclusion Exog⁃enous estrogen may promote EMT process of endometrial cancer cells by activating HIF-1αin hypoxia environment.