| Objective:After hypoxic preconditioning with CoCl2 in Ishikawa cells of endometrial cancer,the expression of HIF-1 alpha in the HIF-1 alpha overexpression group,siRNA-HIF-1 alpha group and the control group were analyzed to explore the role of HIF-1 alpha in the process of epithelial-mesenchymal transformation of endometrial cancer.After hypoxic preconditioning with CoCl2 in Ishikawa cells of endometrial cancer,the expression of HIF-1α and epithelial-mesenchymal transformation-related factors were analyzed in estrogen group to explore the role of estrogen under hypoxia.The interaction between estrogen and HIF-1 alpha in the process of epithelial-mesenchymal transformation in endometrial cancer was explored by comparing estrogen+the HIF-1 alpha overexpression group and estrogen+siRNA-HIF-1 alpha group with the control estrogen group.To further study the mechanism of HIF-lalpha and estrogen on epithelial-mesenchymal transition in endometrial cancer.Methods:Hypoxic preconditioning of endometrial cancer Ishikawa cells was carried out by using cobalt chloride(CoCl2).HIF-lalpha overexpression plasmid and siRNA were transfected into endometrial carcinoma Ishikawa cells by Lipo2000 reagent.After cell cμLture,RNA and protein were extracted for real-time fluorescent quantitative PCR and Western Blot assay.After hypoxic preconditioning of endometrial cancer Ishikawa cells,different groups were carried out:(1)the hypoxic group,HIF-1 alpha over-expression group,siRNA-NC group,siRNA-HIF-1α group;(2)the estrogen group;estrogen+HIF-1 alpha group,the estrogen+siRNA-NC group;estrogen+siRNA-HIF-1 α group;fluorescent PCR and Western Blot were used to observe the expression of HIF-1α and the EMT-related genes in each group of endometrial cancer cells.Results:1.Fluorescence Quantitative PCR Reults:In hypoxic environment,the expression of HIF-1alpha in HIF-1alpha overexpression group and estrogen group increased significantly(P<0.01),while the expression of HIF1alpha in HIF-1α-siRNA group decreased(P<0.01).The expression level of E-cadherin,one of epithelial-mesenchymal transition-related factors,decreased both in HIF-1α overexpression group and estrogen group(P<0.01);while the expression level of E-cadherin in HIF-1α-siRNA group increased significantly(P<0.01).The expression levels of betacatenin,N-cadherin and TGF-beta-1 in HIF-1a overexpression group and estrogen group increased(P<0.01),but decreased in HIF-1a-siRNA group(P<0.01).Compared with the control estrogen group,the expression of HIF-1alpha in the estrogen group+HIF-lα overexpression group increased significantly(P<0.01)and decreased in the estrogen+HIF-1α-siRNA group(P<0.01).The expression level of E-cadherin in estrogen+HIF-1α overexpression group decreased significantly(P<0.01),while E-cadherin in estrogen+HIF-1α-siRNA group increased significantly(P<0.01).The expression levels of beta-catenin,N-cadherin and TGF-beta 1 in the estrogen+HIF-1αoverexpression group increased significantly(P<0.01),while the expression levels in the estrogen+HIF-1α-siRNA group decreased significantly(P<0.01).2.Experimental results of Western Blot:In hypoxic environment,the expression level of HIF-1α in HIF-1α overexpression group was significantly higher than that in control group(P<0.01),but the expression level of HIF-1α-SiRNA interference group was significantly lower(P<0.01);the expression level of E-cadherin in HIF-1α overexpression group was decreased(P<0.05),and the expression level of HIF-1α-SiRNA interference group was significantly lower(P<0.01).The levels of β-catenin(P<0.05),N-cadherin(P<0.01)and TGF-β1(P<0.01)increased significantly in HIF-1α overexpression group and decreased significantly in HIF-1α-SiRNA interference group(P<0.01).Compared with the control estrogen group,the expression of HIF-1α increased in the estrogen+HIF-1α overexpression group(P<0.05),but decreased significantly in the estrogen+HIF-1α-SiRNA interference group(P<0.01);the expression of E-cadherin in the estrogen+HIF-la overexpression group decreased(P<0.05),and in the estrogen+HIF-1α-Si overexpression group(P<0.05).The expression level of RNA interference histone protein increased significantly(P<0.01).The expression levels of beta-catenin(P<0.01),N-cadherin(P<0.01),TGF-beta 1 increased significantly in the estrogen+HIF-1α overexpression group,while the expression levels of beta-catenin(P<0.01),N-cadherin(P<0.05),and TGF-beta 1(P<0.01)decreased significantly in the estrogen+HIF-la overexpression group.Conclusion:1.Real-time fluorescence quantitative RT-PCR and Western blotting showed that the expression of HIF-1α was up-regulated in hypxic group、HIF-la overexpression group and down-regulated in HIF-1α-siRNA interference group.It is suggested that the activation of HIF-1α in hypoxic environment may be involved in the pathogenesis of endometrial cancer,and that hypoxia may participate in the development of endometrial cancer.2.Real-time fluorescence quantitative RT-PCR and Western blotting showed that the expression level of HIF-1α was up-regulated in the estrogen group,indicating estrogen involving in the hypoxia process and up-regulating the expression of HIF-1αa.These resul ts suggest that estrogen plays an important role in the activation of HIF-1α in cancer cells under hypoxia3.Under hypoxia,the expression levels of HIF-1α,E-cadherin,N-cadherin,beta-catenin and TGF-beta 1 in endometrial cancer cells were altered by over-expression and silencing of HIF-1α respectively,suggesting that there may be epithelial-mesenchymal transition in endometrial cancer.On the basis of hypoxia,estrogen intervention was carried out on endometrial cancer cells,and hypoxia-inducible factor HIF-1α was overexpressed and silenced respectively.The changes of the expression level of epithelial-mesenchymal related factors suggest that estrogen may interact with HIF-1α to promote the process of epithelial-mesenchymal transformation of endometrial cancer and promote the occurrence and development of endometrial cancer. |
