TOB1 Modulates The Decidualization Of Human Endometrial Stromal Cells By Notch Pathway

?Background and purpose?Pregnancy begins with successful embryo implantation.There are two prerequisites for embryo implantation: high quality embryo and receptive endometrium.With the continuous development of medical technology and the popularization of assisted reproductive technologies such as in vitro fertilization and embryo transfer(IVF-ET)and preimplantation diagnosis(PGD),the problem of embryo quality is improving day by day.However,the overall success rate of assisted reproductive is still less than 50%,and about two-thirds of the failures are related to endometrial factors.Therefore,endometrial receptivity has become one of the main research hotspots in the field of reproduction.Receptivity refers to the ability to accept embryos,which involves the proliferation,differentiation,apoptosis and other regulatory mechanisms of related cells,and is also closely related to the secretion of a variety of hormones.Tob1(human erb B-2 transcription factor 1)is a member of the antiproliferative protein family BTG / tob.It can affect cell proliferation,differentiation and apoptosis through bmp-smad pathway and MAPK / ERK pathway.The proliferation,differentiation and apoptosis of related cells are closely related to endometrial receptivity,but there is no research report on the relationship between Tob1 and endometrial decidualization.Therefore,this paper aims to study the expression of Tob1 in different stages of endometrium and explore the possible mechanism of its influence on decidualization,hoping to find a new target to improve endometrial receptivity.?Materials and methods?1.Objective to detect the expression of Tob1 in human endometrium at different periods of menstrual cycle.Methods: the endometrial samples of 10 patients who underwent hysteroscopic surgery in our gynecological clinic(different menstrual periods)were collected.The specific stages were confirmed by pathology after surgery.The expression of Tob1 in endometrial proliferation and secretion was detected by Western-blot and QRT-PCR2.Objective to establish an in vitro cell model and further study the changes of Tob1 before and after decidualization and its effect on cell cycle.Methods:E2P4 was used to induce decidualization of endometrial stromal cells(h ESCs).The expression of decidualization markers(IGFBP-1,PRL)was detected by Western blot and immunofluorescence.CCK-8 was used to measure the cell proliferation activity before and after decidualization.The cell cycle changes before and after decidualization were detected by flow cytometry.3.Objective to explore the possible mechanism of Tob1 affecting decidualization.Western blot was used to detect the expression of notch and Tob1 before and after decidualization;In addition,the expression of Tob1 and the change of cell cycle were detected after dapt treatment.?Results?1.The results of QRT-PCR,immunohistochemistry and Western blot showed that the expression level of Tob1 in secretory phase was significantly higher than that in proliferative phase(P < 0.05).2.In order to further verify,the in vitro cell experiment was carried out.After successful decidualization of endometrial stromal cells(h ESCs)induced by E2 and P4,the expression of Tob1 after decidualization was detected by Western blot and immunofluorescence,and the difference was statistically significant(P < 0.05).3.In order to further clarify the effect of Tob1 expression on endometrial cells,CCK-8 test and flow cytometry were used to detect the activity of endometrial cells.It was found that the activity of endometrial cells decreased after decidualization,and the flow cytometry results showed that endometrial cells stagnated in G1 phase.The quantitative analysis of cyclins in G1 phase also showed that endometrial cells stagnated in G1 phase.4.After using si RNA to interfere with Tob1,CCK-8 and flow cytometry showed that interference with Tob1 could restore the proliferation activity of some cells,reduce the number of cells stagnating in G1 phase and increase the number of cells entering S phase,thus affecting the decidualization process.At the same time,the expression of cyclin D1,cyclin E1 and CDK2 in G1 phase were up-regulated after the interference of Tob1,which indicated that Tob1 could make the endometrial cells stagnate in G1 phase and promote their differentiation into decidual cells.When Tob1 was knocked down,the cells could resume proliferation.5.After decidualization,the expression of Notch1 was up-regulated,which was consistent with the expression trend of Tob1.When si RNA successfully knocked down Tob1,the expression of Notch1 decreased.After dapt treatment,the expression of Notch1 decreased,but the expression of Tob1 had no significant change.These results indicate that Tob1 is the upstream regulator of Notch1.Tob1 affects the cell cycle of endometrial cells through Notch1 pathway,which makes them stagnate in G1 phase,thus inhibiting the proliferation of endometrial cells and promoting the decidualization of endometrial cells.?Conclusion?Studies have shown that the expression level of Tob1 in secretory endometrium after decidualization is significantly higher than that in proliferative endometrium.Tob1 plays an important role in the process of decidualization.In vitro experiments further showed that Tob1 may be involved in the regulation of decidualization through Tob1 /Notch cascade,resulting in the arrest of stromal cells in G1 phase.