The Mechanism Of Foxf2 And Smad6 Co-regulation Of Collagen 5A2 Transcription Involved In The Pathogenesis Of Fibrosis In Intrauterine Adhesion

Background and objectiveIntrauterine adhesions(IUA)is a major health problem involving the female reproductive system for women of childbearing age.The pathogenesis of IUA involves decreased or absent endometrial glands,and the endometrial stroma is largely replaced by fibrous tissue,leading to uterine cavity deformation and endometrial fibrosis.Excessive deposition of collagen is the characteristic feature of endometrial fibrosis,but the mechanism undering the production of collagen is still unknown.In our previous study,IUA samples were collected for whole genome expression spectrum chip assay and the result showed the expressions of COL5A2,Foxf2,Smad6 were abnormal in IUA.We predicted there may be presence of transcription factor binding sites for Foxf2 or Smad6 in the promoter region of COL5A2 by online promoter analysis tools.Forkhead box transcription factor F2(Foxf2)is a transcription factor that is widely expressed in mesenchymal tissues and plays an important role in the production of collagen.Smad6 is the downstream mediator of the TGF-?/bone morphogenetic protein(BMP),which can inhibit collagen production through negatively regulating the TGF-? signaling pathway.Therefore,we predicted Foxf2 and Smad6 may co-regulate COL5A2 transcription involved in the pathogenesis of IUA.In the present study,we study the role of Foxf2 and Smad6 involve in IUA fibrosis in vitro and in vivo.Co-immunoprecipitation(Co-IP),chromatin immunoprecipitation(ChiP),and dual-luciferase reporter assays were performed to confirm the interaction between Foxf2 and Smad6 and their regulation of COL5A2 transcription.The result would illuminate the machenism that Foxf2 and Smad6 involved in the endometrial fibrosis and provide a theoretical basis for its prevention and treatment of IUA.Methods and results1.The effect of Foxf2 downregulation or(and)Smad6 upregulation on TGF-?1 induced human endometrial stromal cell line fibrosisMETHODS:TGF-?1 was used to establish a THESCs fibrosis model.si-Foxf2 or(and)pc-DNA3.1-Smad6 were transfected into THESCs before and after the stimulation of TGF-?1 for the purpose to detect the effect of downregulation of Foxf2 or upregulation of Smad6 on TGF-?1 induced fibrosis.qRT-PCR,western blotting performed to examine the expression of COL5A2,COL1A1,FN,and a-SMA.EdU assay was used to detect cell proliferation.Flow cytometric analysis were performed to detect cell cycle distribution.RESULTS:TGF-?1 induced THESCs fibrosis and promoted the expressions of COL5A2?COL1A1?FN.TGF-?1 promoted G0/G1 phase transition into S phase and cell proliferation.Downregulation of Foxf2 or upregulation of Smad6 inhibited TGF-?1 induced fibrosis,and reversed cell cycle changes and cell proliferation induced by TGF-(?1,and combination of downregulation of Foxf2 with upregulation of Smad6 were more effectively to inhibit fibrosis than either of them.2.Foxf2 and Smad6 co-regulate COL5A2 transcriptionMETHODS:immunofluorescence(IF)staining was performed to detect the lacation of Foxf2 and Smad6,and their relation with COL5A2 expression.Co-immunoprecipitation(Co-IP),chromatin immunoprecipitation(ChiP),and dual-luciferase reporter assays were performed to confirm the interaction between Foxf2 and Smad6 and their regulation of COL5A2 transcription.LV-Foxf2 and pcDAN3.1-Smad6 were transfected into HESCs,and qRT-PCR was used to detect COL5A2 expression for the purpose to reveal the relation between Foxf2 and Smad6 on COL5A2 expression.RESULTS:Foxf2 and Smad6 were accumulated in nucleus,Foxf2 promoted COL5A2 expression,Smad6 inhibited Foxf2 induced COL5A2 expression,but cannot completely inbibit.Foxf2 interacted with Smad6,both factors bound to the same promoter region of COL5A2 to regulate its transcription.3.The effect of Foxf2 downregulation or/and Smad6 upregulation on fibrosis in the pathogenesis of rat IUA and pregnancy result for IUA ratMETHODS:Curettage and infection were used to creation of a female rat IUA model.ADV2-Foxf2-1810 and ADV4-Smad6 were injected into uterine wall when creation of IUA model for the purpose of downregulation of Foxf2 and upregulation of Smd6.Hematoxylin/eosin staining(HE)was performed to detect the number of glands in endometrium,Masson trichrome staining was prefprmed to detect fibrosis,and immunohistochemistry was use to detect COL5A2,COL1A1,Foxf2,Smad6 and av?3 expression.Electron microscrope was performed to detect pinopodes in the endometrial epithelium for the purpose to examine the endometrial receptivity.Number of embryo was calculated after intercourse with male rat.RESULTS:Foxf2 downregulation or Smad6 upregulation decrease fibrosis in the pathogenesis of rat IUA,the number of glands in the endometrium was increased,the percentage of fibrosis area was decreased,the proteins expression of COL5A2 and COL1A1 were decreased.Foxf2 downregulation or Smad6 upregulation improved endometrial receptivity,the expression of ?v?3 and pinopodes was increased,and the numbers of embryo was increased.Conclusion:Foxf2 and Smad6 co-regulate COL5A2 transcription involved in the pathogenesis of fibrosis of intrauterine adhesion.Downregulation of Foxf2 or upregulation of Smad6 inhibited fibrosis and improve endometrial receptivity,and increased the numbers of embryo.