Nur77 Promotes Embryo Adhesion Via Transcriptionally Regulating HOXA10

Object:Reduced HOXA-10 expression may contribute to embryo implantation failure,because HOXA 10 is an important regulator of embryo adhesion.It has been demonstrated that the expression of orphan nuclear receptor Nur77 and HOXA 10 is reduced in the endometrium of recurrent implantation failure patients.The aim of this study was to investigate whether Nur77 transcriptionally regulated HOXA 10 to affect embryo adhesion.Methods:1.The transcriptional regulation effect of Nur77 on HOXA10 was determined by Luciferase reporter gene assay,Chromatin immunoprecipitation PCR and Avidin-biotin conjugate DNA precipitation assay;2.Using adenovirus(Ad-Flag-Nur77 or Ad-siNur77)-mediated Nur77 overexpression or knockdown,the effect of Nur77 on HOXA10 expression was detected by Western blot assay in human endometrial cancer cell line-Ishikawa cell;3.6-MP(Nur77 activator)and JAR spheroid attachment assay(human placental villous carcinoma cells-JAR spheroid adhere to Ishikawa cells)were performed to analyze the role of HOXA10 in mediating embryo adhesion regulated by Nur77.Results:The luciferase dual reporter gene,chromatin immunoprecipitation PCR and avidin-biotin conjugate DNA precipitation assay demonstrated that Nur77 bound directly to the NBRE response element AAAGGTCA(-3075/-3067)of the HOXA10 promoter region and then promoted HOXA10 transcription in Ishikawa cells.Ad-Flag-Nur77-mediated Nur77 overexpression promoted the level of HOXA10 protein in a concentration-dependent manner in Ishikawa cells,and increased the adhesion efficiency of JAR spheroids to Ishikawa cells(57±4.2%vs 34±5.5%,p<0.05,vs Ad-LacZ);When Ad-siNur77 adenovirus disturbed endogenous Nur77 expression of Ishikawa cells,HOXA10 expression was effectively inhibited,meanwhile the adherence between JAR spheroids and Ishikawa cells was significantly decreased(24.3±3.7%vs 38±3.0%,p<0.05,vs Ad-CTL).When the endogenous HOXA10 expression was knockdown by CRISPR-Cas9-adenovirus in Ishikawa cells,the role of Nur77 promoting JAR spheroids attachment was significantly inhibited.Nur77 agonist 6-MP promoted embryo adhesion(62.3±1.8%vs 38.311.4%,p<0.0001,vs Ad-CTL).Conclusion:This study was the first to find that orphan nuclear receptor Nur77 promoted embryo adhesion through transcriptionally regulating HOXA10 expression.It suggested that orphan nuclear receptors Nur77 and its agonist 6-MP were expected to provide a new treatment bassis for patients who suffered from recurrent implantation failure caused by embryo adhesion dysfunction in assisted reproduction.