| Objective:The decidualization of human endometrial stromal cells is critical for the successful establishment and maintenance of pregnancy and involves extensive cell proliferation and differentiation.The purpose of the present study was to explore the function and mechanism of β-catenin in the decidual development of human endometrial stromal cells.Methods:1.We collected human endometrial tissues from mid-proliferative to mid-secretory of the menstrual cycle and decidual tissues of normal early pregnancy women,and analyzed the β-catenin expression by immunohistochemistry staining;2.Human endometrial stromal cell line(HESCs)were induced to be decidualized in vitro.Western blot(WB)and immunofluorescence(IF)techniques were used to detect the expression and localization of β-catenin during decidualization;3.Wnt and β-catenin transcription inhibitor iCRT3 were added to induced decidualization in vitro,and morphological changes of decidualization cells were observed under light microscope.The mRNA and protein levels of insulin like growth factor binding protein 1(IGFBP-1),prolactin(PRL)and transcription factors FOXO1,PR and C/EBPβwere detected by quantitative real-time PCR(QRT-PCR)and WB;4.The recombinant plasmid CTNNB1-APEX2-NLS(The protein encoded by CTNNB1 gene is β-catenin.APEX2,engineered ascorbate peroxidase;NLS,Nuclear Localization Sequence)was constructed,and the feasibility of the recombinant plasmid was verified by WB,IF and Immunoprecipitation(IP);5.The control APEX2-NLS and CTNNB1-APEX2-NLS lentivirus were infected with the proliferation and differentiation stages of HESCs.After 48 hours,biotin phenol(BP)and hydrogen peroxide(H2O2)were added to label the adjacent proteins.The biotin protein was purified by streptavidin magnetic beads,and the purified proteins were detected by mass spectrometry;6.The interaction between β-catenin and the candidate protein was verified by mass spectrometric data analysis.Results:1.β-catenin was widely expressed in the gland epithelium and the stromal cells at different phases of menstrual cycle and decidual tissues,and was localized in the nucleus;2.β-catenin was expressed at 2,4 and 6 days undergoing decidualization.Nuclear and cytoplasmic separation assay,and immunofluorescence assay showed that β-catenin was mainly localized in the nucleus.After β-catenin inhibitor was added,the mRNA and protein levels of IGFBP-1 and PRL were significantly decreased.The mRNA and protein levels of FOXO1,PR,C/EBPβ were lower than those in the control group;3.The recombinant plasmid CTNNB1-NLS-APEX2 was successfully constructed.Cells were confirmed by immunofluorescence to be infected by CTNNB1-NLS-APEX2 lentivirus,and the biotinylated protein was co-localized with it.WB confirmed that the surrounding proteins were labeled by biotinylation after lentivirus infection.IP experiment was used to enrich and purify the biotinylated protein,which further confirmed the feasibility of the system in HESCs;4.The results of mass spectrometry showed that compared with the control group,there were 530 proteins in the proliferation stage and 687 proteins in the differentiation stage,respectively.These molecules are mainly involved in the process of metabolism,localization by the GO biological information;5.HOXA10 was selected from the mass spectrum data,and the in vitro immunoprecipitation experiment verified that there was no strong interaction relationship between HOXA10 and β-catenin in the decidualization.Conclusion:1.β-catenin was expressed in the mid-proliferative,mid-secretory and early decidual stages of the human menstrual cycle;2.β-catenin was expressed in the differentiation stage of HESCs,and was involved in regulating the decidualization of human endometrium by affecting the expression of FOXO1,PR,and C/EBPβ;3.CTNNB1-NLS-APEX2 could biotinylate surrounding proteins,confirming the feasibility of this system in HESCs.And,IP experiments had confirmed that β-catenin and HOXA10 had no strong interaction. |
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