The Function And Molecular Mechanism Of FFAR4 In Endometrial Cancer

Objective : Free fatty acid receptor 4(FFAR4)is widely involved in cell pathophysiological processes such as inflammation regulation,hormone secretion regulation,glucose and lipid metabolism,and cell proliferation,which is closely related to a variety of human cancers.However,the involvement of FFAR4 in uterine corpus endometrial carcinoma(UCEC)remains unclear.This study aims to explore the role and mechanism of FFAR4 in endometrial carcinoma,and provide a potential new target for the treatment of endometrial carcinoma.Methods : The data of UCEC patients were downloaded from the TCGA-UCEC dataset of the TCGA database for comparison.The expression of FFAR4 in endometrial carcinoma tissues and adjacent normal tissues was analyzed by the Mann-Whitney U test.The expression level of FFAR4 on overall survival(OS)and disease free survival(DFS)of UCEC patients was analyzed by GEPIA database.P <0.05 was considered to be statistically significant.Eighty-three patients pathologically diagnosed as UCEC and underwent radical surgical resection in the Affiliated Hospital of Qingdao University from 2019 to 2021,were collected in this study.The relationship between FFAR4 expression level and clinicopathological data was analyzed by immunohistochemical staining.The protein expression levels of FFAR4 in four endometrial carcinoma cell lines Ishikawa,RL95-2,HEC-1-A and AN3 CA were detected by Western Blot.Cell experiments(cell proliferation assay,flow cytometry and Transwell migration and invasion assay)were conducted to study the effects of FFAR4 on proliferation,apoptosis,migration and invasion of endometrial carcinoma cells.Western blot was used to detect the changes of β-catenin cytoplasmic and nuclear protein levels and c Myc protein expression levels after FFAR4 activation or antagonism in endometrial carcinoma cells.Results : The analysis of UCEC patients included in the TCGA-UCEC dataset showed that the expression of FFAR4 in endometrial carcinoma was higher than that in adjacent normal tissues(P = 0.049,95 % CI : 0-1.307).The disease free survival of patients with high FFAR4 expression in GEPIA database was significantly higher than that of patients with low FFAR4 expression(P = 0.0038,HR = 0.31).Immunohistochemical staining showed that FFAR4 was highly expressed in endometrial carcinoma compared with adjacent tissues.The high expression of FFAR4 was correlated with clinicopathological stage(P = 0.008)and triglyceride(P= 0.025)level.Cell proliferation assay in vitro showed that TUG891-induced FFAR4 activation inhibited endometrial carcinoma cells proliferation(P < 0.05)and promoted apoptosis in Ishikawa and RL95-2 cells(P < 0.05).Transwell assay in vitro showed that TUG891 could inhibit the migration and invasion of endometrial carcinoma cells in Ishikawa and RL95-2 cells(P < 0.05).Western Blot showed that c Myc protein expression which was regulated by WNT-β-catenin pathway changed with the change of FFAR4(P < 0.05).Conclusion : 1.FFAR4 is significantly overexpressed in endometrial carcinoma tissues,and patients with low FFAR4 expression have poor prognosis.High FFAR4 expression was correlated with clinicopathologic staging and triglyceride level.2.The activation of FFAR4 could inhibit the proliferation of Ishikawa and RL95-2 cells and promote the apoptosis of Ishikawa cells in vitro.The inhibition of FFAR4 had no significant effect on the proliferation and apoptosis of endometrial carcinoma cells.3.The activation of FFAR4 can inhibit the migration and invasion of Ishikawa and RL95-2 cells,and the inhibition of FFAR4 has no significant effect on the migration and invasion of endometrial carcinoma cells.4.Mechanistically,the function of FFAR4 in endometrial carcinoma cells could affect the protein expression level of c Myc by WNT-β-catenin pathway,possibly affecting cell apoptosis,cell migration and invasion ability through this pathway.