The Role And Mechanism Of Hippo Signaling Pathway In Uterine Adhesions And Thin Endometrium

BackgroundInfertility is a global health problem affecting approximately 15-20%of couples of reproductive age.Despite the rapid development of assisted reproductive technologies in recent decades,embryo implantation failure still occurs in most IVF-ET cycles(60-70%).A good endometrial receptivity is necessary for a normal embryo to be able to implant successfully.Transvaginal ultrasound measurement of endometrial thickness is a simple,practical and non-invasive prognostic indicator for assessing endometrial receptivity.Thin endometrium can be caused by intrauterine operations such as curettage and uterine adhesions,which can affect endometrial receptivity and prevent embryo implantation and development,resulting in decreased implantation rate and clinical pregnancy rate.Currently,the clinical definition of thin endometrium is endometrial thickness ≤7mm on HCG day.Thin endometrium is pathologically characterized by slow growth of glandular epithelium and poor proliferation of endometrium.To date,the pathogenesis of thin endometrium is unclear.The Hippo signaling pathway is a recently discovered pathway closely related to proliferation,which plays an important role in controlling organ volume,stem cell function,tissue regeneration and tumorigenesis.The core molecule of the Hippo signaling pathway,YAP,promotes cell proliferation and inhibits cell apoptosis by forming a complex with TEAD in the nucleus to increase the expression of the downstream growth factor CCN and the apoptosis inhibitor BIRC.Jasplakinolide(JASP),a natural cyclic peptide derived from sponges,is a potent inducer of actin polymerization.The cytoskeleton is a protein-fiber lattice structure in eukaryotic cells and consists of three main constituents:microfilaments,microtubules and intermediate fibers.Among them,microfilaments consist of actin in both monomeric and polymerized states,and regulate many physiological activities of cells,such as stress fiber formation,cell attachment,migration,apoptosis,material transport,transmembrane information transfer,and receptor aggregation.Previous studies have shown that JASP can induce the conversion of the monomeric form of actin(G-actin)to the polymeric form of actin(F-actin),thus promoting the activation of YAP,which enters the nucleus and induces the expression of growth factor CCN and apoptosis inhibitory gene BIRC5,thereby promoting follicle development,and is a potential therapeutic-agent for the treatment of polycystic ovary syndrome and other ovarian diseases.However,the role of JASP and Hippo signaling pathways in the endometrium is still unclear.ObjectiveThe aim of this study was to clarify the role of Hippo signaling pathway in the physiological process of normal endometrium and the pathogenesis of uterine adhesions and thin endometrium and the mechanisms involved,as well as whether JASP can regulate the repair process of endometrial cells by mediating Hippo signaling pathway.Methods1.Normal endometrial tissues of proliferative and secretory phases and decidual tissue were collected,and immunohistochemistry was used to detect the expression differences of YAP protein(the core molecule of Hippo pathway)in these three periods;qRT-PCR was used to detect the changes of LATS,YAP,TEAD,CCN and BIRC(the related molecules of Hippo pathway)in different periods;Western blot was used to detect the expression differences of Hippo pathway core molecules YAP and phosphorylated YAP(p-YAP)protein in these three periods.2.Collect the endometrial tissue of patients with uterine adhesions and thin endometrium as the experimental group,and the normal endometrial as the control group.Immunohistochemistry was used to detect the difference of YAP protein expression between thin endometrium and normal endometrium;qRT-PCR was used to detect the difference of Hippo pathway related molecules LATS,YAP,TEAD,CCN and BIRC in the endometrium of the two groups;Western blot was used to detect the difference of YAP and p-YAP protein expression between the experimental group and control group.3.Endometrial adenocarcinoma cell line(Ishikawa cells)and human primary endometrial stromal cells(HESC)were selected,and Ishikawa cells and HESC were treated with Hippo pathway inhibitor Jasplakinolide(JASP)to detect the scratch healing ability of the cells by cell scratch assay;JASP was detected by qRT-PCR Changes in mRNA levels of Hippo pathway-related molecules in cells after treatment with JASP.Results1.YAP,the core molecule of the Hippo pathway,is expressed in both endometrial epithelial cells and stromal cells,and there were significant differences between different periods.In stromal cells,the expression of YAP decreased significantly from proliferative phase to secretory phase,and increased from secretory phase to meconium;in epithelial cells,the expression of YAP decreased gradually from proliferative phase to secretory phase,and there was no statistical difference between meconium and secretory phase.The mRNA levels of YAP,LATS1,TEAD1,CCN1,CCN2,CCN4,and BIRC5 were significantly higher in proliferative phase compared with those in secretory phase and decidual tissues(P<0.05).2.Immunohistochemical results showed that YAP expression was significantly decreased in both stromal and epithelial cells of thin endometrium patients with a history of previous uterine adhesions compared with normal endometrium.The mRNA levels of YAP,TEAD1,CCN2,and BIRC5 were significantly decreased in thin endometrium compared with normal endometrium,and the mRNA levels of YAP,LATS1,TEAD1,CCN1,and BIRC5 were significantly decreased in endometrium with uterine adhesions.Western blot results showed that in thin endometrium and endometrium with uterine adhesions,YAP and p-YAP protein expression levels were significantly decreased(P<0.05).3.After treatment of Ishikawa and human primary endometrial stromal cells with JASP,the scratch healing ability of both cells increased significantly;the mRNA levels of YAP and BIRC5 in JASP-treated Ishikawa cells gradually increased(P<0.05).Conclusion1.Hippo signaling pathway may be involved in the proliferation process of normal endometrium.During the proliferative phase of normal endometrium,Hippo signaling pathway is inhibited,resulting in increased YAP dephosphorylation and nuclear localization.YAP promotes the proliferative process of endometrium by binding to genes such as TEAD,CCN and BIRC in the nucleus.2.Hippo signaling pathway may be involved in the pathogenesis of intrauterine adhesions and thin endometrium.In intrauterine adhesions and thin endometrium,Hippo signaling pathway is activated,resulting in decreased nuclear localization of YAP,which inhibits its binding to downstream target genes,thus affecting the proliferative process of endometrium.3.JASP may promote the repair process of endometrial cells by upregulating YAP.