Fuction Of Bile Acid Nuclear Receptor FXR In Ovarian Cancer Cells

BackgroundOvarian cancer(OC)is the female reproductive system tumor with high mortality rate.The traditional of ovarian cancer treatment methods are not satisfactory.Finding new therapeutic targets is of great significance for the treatment of ovarian cancer.STAT3 signal pathway plays an important role in the differentiation,growth and apoptosis of cells.Activation of STAT3 signal pathway has been detected in ovarian cancer,and knocking down of STAT3 can inhibit the growth and migration of ovarian cancer.Therefore,the study of STAT3 signaling pathway in ovarian cancer is of great significance.Farnesoid X receptor(FXR)is a bile acid nuclear receptor that affects many physiological functions of the body.Endometriosis is a cause of ovarian cancer,and FXR can treat endometriosis by regulating STAT3 signaling.But the role of FXR in ovarian cancer has not been reported.We conducted this study to investigate the specific role of FXR in ovarian cancer cells.ObjectivesThis paper reveals the role of FXR in ovarian tissue and human ovarian cancer cells and its molecular mechanism through in vitro and in vivo experiments.MethodsIn vivo experiment:Model ?:FXR KO female mice and C57BL/6J wild-type female mice,9-10 weeks old,were selected to ensure complete sexual maturity with hormone-free feed feeding and to avoid individual differences in mating.Serum samples were collected,RNA and tissue proteins in ovarian tissue were extracted,and the specific effects of FXR knockout on mouse ovary and estrogen levels in vivo were investigated by real-time quantitative PCR,ELISA and Western blotting experiments.Model ?:Allogeneic tumor transplantation experiments were performed on typical human OC cell line SKOV3 with high tumor-forming rate.BALB/c mice has no thymus and is T cell immunodeficiency.The well-cultured SKOV3 cells were resusfended in a volume of 150?L in a PBS solution and implanted in the axilla of the nude mice with 5×10 ~6 cells per mouse,and tumor formation was observed in 5th day.The mice were randomly divided into two groups,and the drugs were injected intraperitoneally.The experimental group was intraperitoneally injected with FXR agonist GW4064,which was dissolved in DMSO(6 mg/mL),and the dose injected was 30 mg/kg body weight.The control group was injected with the same amount of DMSO,and the tumor size was measured using a vernier caliper.Two days for one cycle of administration,the drug was simultaneously measured for tumor size and administered for 8 cycles.At the end of the last administration,the nude mice were sacrificed,the tumor was removed for immunohistochemical staining of Ki67 to evaluate the degree of tumor proliferation.Allogeneic tumor transplantation experiments to explore the effect of intraperitoneal injection of FXR agonists on tumors of allograft ovarian cancer.In vitro experiments:Phenotypic experiment:Human OC cell line SKOV3 and OVCAR8 were cultured for experiments.The experimental groups were divided into GW4064 experimental group and DMSO control group.The MTT assay was firstly performed.In SKOV3 cells,the experimental group FXR agonist was used at a concentration of 1?M and 2?M,and the concentration of FXR agonist were 0.5?M and 1?M in OVCAR8 cell.To investigate the effect of FXR agonists on the proliferation of ovarian cancer cells by recording the OD values at different time points after agonist treatment.Secondly,the cell scratch test was carried out.The experimental group and agonist concentration were the same as those of the MTT test.The degree of cell scratch healing in the experimental group and the control group at different time points was photographed,and the effect of FXR on the migration of ovarian cancer cells was explored.Finally,SKOV3 cells were treated with FXR agoinst for 48 hours,and all cells including dead cells were recovered,and were stained with Annexin-V-FITC/PI.The effect of FXR on apoptosis of human OC cells was examined by double staining.Mechanism study:The appropriate concentration of GW4064 was selected,according to the phenotypic experiment results.The two?M of GW4064,which was used to treat SKOV3 cells,while OVCAR8 cells were trested with 1?M GW4064.Firstly,to inverstigate the gene regulation of FXR agoinst on OC cells,cells were treated with GW4064 for 24 h,the RNA of living cells was extracted,and relevant inflammatory genes and apoptotic genes were measured to explore the regulation of FXR agonists on human OC cell genes.Secondly,OC cells were treated with GW4064 for 24h then treated with human IL-6(10 ng/ml)for 6h to activate STAT3 signaling pathway in cancer cells.Then,we extracted total cellular protein for Western blotting and detected the level of phosohorylated STAT3(Tyr705).The level of phosohorylated STAT3 protein(Tyr705)in cancer cells was detected.The effect of FXR activation on STAT3(Tyr705)phosphorylation in cytoplasm was observed,and the mechanism of FXR on STAT3 signaling pathway in human ovarian cancer cells was explored.Verification experiment:Small interfering RNA targeting was FXR,transfected into human ovarian cancer cells and the knockdown efficiency of FXR was measured.The siRNA with the most obvious knockdown effect was used to perform MTT colorimetric assay to verify the cell proliferation after FXR knockdown.Results1.In ovarian tissues of FXR KO mice,the expression levels of IL-2,IL-4,INF-?and other inflammatory genes were higher than those in WT mice;and the phosphorylation levels of STAT3(Tyr705)protein was also significantly higher than that in WT mice.The level of estradiol in the serum of FXR KO mice was lower than that in wild type mice.(P<0.05)2.In xenograft model,the growth rate,tumor volume and proliferation index of tumor tissue in the experimental group were significantly lower than those in the control group.(P<0.05)3.MTT colorimetric assay and cell scratch test showed that activation of FXR expression in OC cells could significantly inhibit the proliferation and migration of cancer cells.At the same time,FXR could promote the apoptosis of OC cells.(P<0.05)4.Activated FXR can inhibit the inflammatory genes Cox-2,MMP2,IL-6,IL-4,and MCP-1(P<0.05),up-regulate the proapoptotic genes p53,p21,p27(P<0.05),Caspase3,c-Myc,and Bax(P<0.01).FXR activation could inhibitied of IL-6 induced p-STAT3(Tyr705)protein levels in OC cells.(P<0.05)5.Silencing FXR by its specific siRNA can promote the growth of OC cells.(P<0.05)Conclusions1.Knockout of FXR can activate the phosphorylation of STAT3(Tyr705)protein and mRNA levels of some inflammatory factors in mouse ovarian tissue,and reduce serum estradiol levels.2.FXR agonist GW4064 can negatively regulate the expression of inflammatory cytokines and up-regulate the mRNA level of pro-apoptotic genes by inhibiting the STAT3 signaling pathway in human ovarian cancer cells,which can cause apoptosis of ovarian cancer cells and inhibit proliferation and migration of OC cells.3.FXR agonist GW4064 can inhibit the growth of allograft ovarian cancer cells through peritoneal absorption.