The Study Of Transcription Factor Hoxa11 Promoting Plasma Membrane Transformation Of Emdometrial Epithelial Cells By Regulatingin PD-L1 Expresion In Dairy Cows

Although China’s dairy cattle industry has developed well in recent years,the problem of dairy cow reproductive obstacles still seriously restricts the development of dairy industry.Clinically,the loss of early embryos leads to abortion and even infertility of the cows,causing substantial economic losses to the breeding industry.Emerging studies have shown that low endometrial receptivity is considered to be the most critical factor in embryo implantation failure and loss.Evidence shows that plasma membrane transformation of endometrial epithelial cells in the early pregnancy under the stimulation of various hormones,can provid a suitable physiological environment and structural basis for embryo implantation,which is also a sign of the successful establishment of endometrial receptivity,and the imbalance of this process will directly lead to the failure of embryo acceptance and thereby causing embryo loss.HoxA11 has been shown to play a vital role in embryo implantation,and the stimulation of estrogen and progesterone can remarkably increase the expression of HoxA11.Also,as a transcription factor,HoxA11 can target other genes to regulate the development of the endometrium.As a ligand of PD-1,PD-L1 owns the property of mediating immune tolerance.However,the function of PD-L1 as a separate gene still deserves further exploration.In cancer-related studies,it has been reported that PD-L1 can promote epithelial-mesenchymal(EMT)transformation of cancer cells through the PI3K/AKT signaling pathway and promote cell migration,which is highly similar to the plasma membrane transformation of endometrial epithelial cells in early pregnancy.Therefore it is interesting to explore whether PD-L1 can also get involve in transforming the plasma membrane of endometrial epithelial cells in early pregnancy.This project aims to elucidate the molecular mechanism of HoxA11 and PD-L1 in the transformation of plasma membrane of bEECs in early pregnancy,which has important scientific significance and clinical value for improving the C of dairy cows.The results can be summarized as follows:1.Plasma membrane transformation in the endometrium of dairy cows in early pregnancyH&E staining and SEM were used to confirm the suitable samples we collected.Subsequently,the expression level of E-cadherin/N-cadherin,a marker of endometrial plasma membrane transformation,was detected by IF and IHC assays,and it was found that the expression of E-cadherin in endometrial epithelial cells in the early stage of pregnancy was significantly reduced,while the expression of N-cadherin was significantly increased,indicating that the endometrium undergoes plasma membrane transformation in early pregnancy.In addition,q RT-PCR results showed that the expression of endometrial receptivity marker,HoxA10 and ITGB3 genes,were significantly increased in early pregnancy,suggesting the close relationship between endometrium plasma membrane transformation and the endometrial receptivity establishment during the stage of embryo implantation.2.The hormone stimulation can promote both the expression of HoxA11 and PD-L1 and the plasma membrane transformation of bEECsIn vitro experiments,when bEECs were treated with E2,P4 and IFN-τ,the expression of HoxA11 and PD-L1 were both increased,and the expression of plasma membrane transformation markers N-cadherin and Vimentin proteins were also upregulated.At the same time,the transcription levels of epithelial polarity-related gene(par3,LLGL2)and tight junction molecules(ZO-1,occludin)were decreased under the combined treatment of E2,P4 and IFN-τ,suggesting a decrease in polarity and tight junction of cells,which was consistent with the marker event of plasma membrane transformation of epithelial cells.3.HoxA11 and PD-L1 can promote plasma membrane transformation of bEECsHoxA11 and PD-L1 lentiviral interference vectors p CD513B-U6-sh HoxA11 and p CD513B-U6-sh PD-L1 were designed and constructed,and HoxA11 and PD-L1 knockdown cell lines were constructed successfully respectively.It was found that under the treatment of E2,P4 and IFN-τ,HoxA11 or PD-L1 gene knockdown could inhibit the plasma membrane transformation of bEECs,which was manifested as high expression of the epithelial polarity-related gene(par3,LLGL2)and tight junction molecules(ZO-1,occludin),a decrease of mesenchymal cell marker proteins N-cadherin and Vimentin,increase of epithelial cell marker protein CK18 and decrease of cell migration rate.It was proved that HoxA11 and PD-L1 could play an essential role in the process of plasma membrane transformation of dairy cow’s endometrium in early pregnancy.4.Transcription factor HoxA11 can regulate the expression of PD-L1JASPAR suggests that the transcription factor HoxA11 may promote the transcription and expression of the PD-L1 gene by targeting its promoter.All the possible binding sites in the PD-L1 promoter region that may interact with the transcription factor HoxA11 were listed.In this project,we chose the top three binding sites with the highest scores and validated them using a dual luciferase reporter assay.Finally,it was proved that the transcription factor HoxA11 coulld promote the expression of the PD-L1 gene,and the binding site 2(TCTTGTAAAAGA)located in the PD-L1 promoter region might be the target sequence of the transcription factor HoxA11.5.PD-L1 regulates endometrial remodeling through PI3K/AKT signaling pathwayThe String database shows that PD-L1 can interact with PI3K/AKT signaling pathway.Western blot and IF experiments were used to detect the expression of PI3 K and AKT proteins,and found that PI3K/AKT signaling pathway can be activated under the treatment of hormones,but when PD-L1 is knocked down,PI3 K and AKT proteins cannot be phosphorylated under the treatment of hormones,which suggested that PD-L1 can promote plasma membrane transformation of the endometrium in early pregnancy through the PI3K/AKT signaling pathway.In summary,this study proved that estrogen,progesterone and IFN-τ can increase the expression of transcription factor HoxA11,and HoxA11 can up-regulate the expression of PD-L1 gene and promote the dynamic changes of bEECs in early pregnancy and therefore establishing a suitable environment for embryo implantation.