SOX4 Facilitates PR Protein Stability And FOXO1 Expression Conducive For Human Endometrial Decidualization

Background:Normal decidual differentiation of endometrial stromal cells plays an important role in embryo implantation,embryo development,and pregnancy.Abnormal decidualization can lead to repeated pregnancy failure,such as repeated implantation failure,repeated biochemical pregnancy,recurrent spontaneous abortion,fetal growth restriction,placental implantation and other pregnancy-related diseases.Although there are many clinical treatment methods for repeated pregnancy failure,including immunotherapy,vasoactive drug therapy,traditional Chinese medicine,intrauterine infusion of h CG,and so on,which have certain effect.But there are still a considerable number of patients with unsatisfactory efficacy.These suggest that the potential molecular regulation of endometrial decidualization is unclear.SOX family are a conserved group of transcriptional regulators that mediate DNA binding through a highly conserved high-mobility group(HMG)domain.Accumulating evidence demonstrates that cell fate and differentiation in major developmental processes are controlled by SOX family.In our study,we found SOX4 was the most abundant SOX family member in stroma cell of human and mouse.Although evidences suggest that SOX4 plays an important role in proliferation,differentiation and epithelial mesenchymal transformation.It has been reported that SOX4 expression was significantly induced in stromal cells after decidualization.It is speculated that SOX4 may play an important role in the process of stromal cell decidualization.Objective: to explore new molecular mechanisms for the regulation of decidualization in order to elucidate clinical diseases related to decidualization.Methods: First,human endometrium specimens were used to detect whether SOX4 changed with the changes of sex hormones in normal menstrual cycles.Then the CRISPR/Cas9 technique was then used to establish knockout SOX4 cell lines.The relationship between SOX4 and deciduation-related molecules PRL,IGFBP1,FOXO1 and PR was determined by q PCR,Western blot(WB),chip-q PCR,IP,RNA-seq,PR-Immunoprecipitation-Mass Spectrometry(PR-IP-MS),protein stability test and ubiquitination test.Finally,clinical samples were taken from endometriosis patients who had failed repeated implantation and endometrium of normal parturient mothers to verify the expression of SOX4 and these deciduation-related molecules.Results: Few SOX4 was observed in proliferative phase,its expression was progressively increased in the nucleus of stromal cell of P4-dominant early,middle,and late secretory phases,as well as in the glands.We further verified SOX4 responsed to Progesterone hormone and was regulated by progesterone–progesterone receptors.Then,using CRISPR/Cas9 to knock out(KO)SOX4 gene,it was found that the expressions of decidualization markers PRL and IGFBP1 decreased significantly compared with wild type(WT)after knocking out SOX4 gene.To dissect out the downstream of SOX4 in decidualization,we found SOX4 directly regulated FOXO1 transcription and SOX4 and P300 physically interacted with each other to promotes histone H3 acetylation enrichment of FOXO1 promoters.We also indicated that SOX4 regulated IGFBP1 mainly through transcriptional activation FOXO1.It is interesting that knocking down SOX4 did not affect the m RNA level of PR,but the protein level of PR.Through protein stability experiment and ubiquitination experiment,it was found that SOX4 regulated the stability of PR protein by ubiquitination proteasome.By RNA-seq sequencing(after SOX4 knockout)and IP-mass spectrometry of PR antibody,the E3 ubiquitin ligase HERC4 of PR was screened out.Using h ESCs and 293 T cells,SOX4-mediated ubiquitination and degradation of PR by regulated E3 ubiquitin ligase HERC4.In endometrial stromal cells during the secretory phase of endometriosis patients who had failed repeated implantation,the expression of SOX4 protein was low,along with the expression of PR,FOXO1,and IGFBI proteins.Finally,clinical samples were used to verify the low expression of SOX4 protein,as well as the low expression of PR,FOXO1 and IGFBI protein in endometrial stromal cells during the secretory phase of endometriosis patients with repeated failed implantation.Conclusion: This study found that SOX4 is a novel modulate of stromal cells of human endometrium decidualization and associated with decidual-related diseases.Abnormal SOX4 may be a potential cause of repeated implant failure during in vitro fertilization(IVF)for endometriosis.