Estrogen Related Receptor γ Promotes Invasion And Metastasis Of Endometrial Cancer Via Targeting S100A4

Part 1.The expression and relationship of S100A4、E-cadherin and Vimentin in endometrial cancerObjective:To investigate the expression profile of S100A4,E-cadherin and Vimentin in endometrial cancer(EC),and to study the role of S100A4 on the expression of E-cadherin and Vimentin in EC.Methods: Immunohistochemistry(IHC)were applied to detect the expression of S100A4、E-cadherin and Vimentin in b enign endometrium(n=15)and endometrial cancer(n=50)tissues.Short hairpin RNA(sh RNA)targeting S100A4 was constructed to silence the expression of S100A4.Western blotting(WB)and immunofluorescence(IF)were adopted to analyse the changes of E-cadherin and Vimentin expression,and morphological transform of cytoskeleton.Results: 1.The expression of S100A4 and Vimentin in glandular epithelial ce lls of EC were mainly located in cytoplasm and nucleus,and their expression was significantly positively correlated(correlation coefficient r = 0.6862,P < 0.0001).While,E-cadherin was mainly located in the membrane and cytoplasm of the epithelial cells,which was negatively correlated with the expression of S100A4(correlation coefficient r =-0.5349,P < 0.0001).2.Silence the expression of S100A4 in HEC-1B cells resulted in the disappearance of cell polarity and morphology changed to epithelial phenotype.In addition,the protein level of E-cadherin was increased and the level of Vimentin was decreased.Conclusion: The role of S100A4 in EC migration and invasion were proved previously.The current studies showed the regulation of Vimentin and E-cadherin by S100A4 in EC,respectively.These results indicated that S100A4 might trigger the progession of EC through epithelial-mesenchymal transition(EMT).Part 2.Expression of ERRγ in endometrial cancer and its regulatory mechanism on S100A4Objective:To clarify the expression profile of ERRγ in EC and its correlation with the expression of S100A4.To investigate the regulatory effects of ERRγ on the expression of S100A4 in EC cells.Methods: qRT-PCR were used to detect the m RN A levels of ERRγ and S100A4 in benign endometrium(n=20)and endometrial cancer(n=20)fresh tissues.IHC were used to analysis the location of ERRγ and S100A4 in EC tissues.Lentivirus vector containing S100A4 gene and plasmid containing ERRγ sh RNA were constructed.Western blotting and qRT-PCR were employed to detect the expression of ERRγ,S100A4 and E-cadherin.Dual luciferase reporter assay were applied to analyse the impact of ERRγ on S100A4 gene promoter activation.Results: 1.The m RNA and protein levels of ERRγ in EC were significantly higher than those in normal endometrial tissues(P = 0.0175).There was a positive correlation between ERRγ and S100A4 transcription level in EC(correlation coefficient r = 0.557,P = 0.01).2.Upregulation of ERRγ led to the up-regulation of S100A4 and down-regulation of E-cadherin in HEC-1A and AN3 CA cells.O n the contrary,downregulation of ERRγ resulted in the down-regulation of S100A4 and up-regulation of E-cadherin in HEC-1B cells.Three sites(368-377 bp,639-648 bp and 731-740bp)before transcription start site were predicted ERRγ binding parts on S100A4 promoter,and the 656-784 bp region was the main active section which ERRγ exerted its function.Upregulation of ERRγ can enhance the S100A4 promoter transcriptional activity.Conclusion: The expression of ERRγ in endometrial carcinoma was positively correlated with the expression of S100A4,and ERRγ could activate the promoter of S100A4 gene which indicated that ERRγ may be an important upstream regulator of S100A4 in EC.However,whether the effect of ERRγ on the biological function regulation of EC cells were mediated by S100A4 remains to be elucidated.Part 3.ERRγ promote invasion,migration and in vivo tumor growth,metastasis via S100A4 in endometrial cancerObjective: To investigate the role of S100A4 on ERRγ induced endometrial cancer cell invasion,migration and in vivo tumor growth.Methods: Transwell assay was used to analyze the impact of ERRγ expression and synchronously restored the expression of S100A4 in EC cells migration and invasion capacity.The 4-6 week female BALB/c nude mice were inoculated with EC cells under the right shoulder to establish the subcutaneous xenograft model.After the formation of the tumor,the volume of the tumor was measured and recorded every 7 days.Twenty-four days after inoculation,nude mice were sacrificed,the tumor was removed,and the expression of ERRγ and S100A4 were detected by IHC.EC cells were injected to BALB/c nude mice through vena caudalis,the nude mice were executed 42 days later and the lung and liver were extracted,the number of metastatic tumor were indicated by hematoxylin and eosin staining.Results: 1.Upregulation of ERRγ expression enhanced the migration and invasion ability of HEC-1A and AN3 CA cells,and restoration of S100A4 expression prevented the enhanced migration and invasion capacity in these cells induced by stable over-expression of ERRγ.Conversely,down-regulated the expression of ERRγ in HEC-1B cells resulted in decreased cell migration and invasion capacity,while restoration of S100A4 could also restore the cell invasion and migration ability of HEC-1B cells.2.The growth rate of tumors in ERRγ overexpression group was obviously higher than that of control group,and the tumor weight of ERRγ overexpression group was also significantly higher than that of the control group(1.22 ± 0.53 g vs 0.27 ± 0.15 g,P <0.01).IHC showed that the levels of S100A4 in the ERRγ stable expression group were higher than those in the control group.3.The number of metastatic tumor in ERRγ group were significantly higher than that of the control group(P= 0.0109).Conclusion: ERRγ promoted migration,invasion and tumor growth,metastasis of EC mainly through S100A4 signaling pathway.