The Relationship Between The Phosphorylation Level Of CacyBP/SIP And The Degradation Level Of β-catenin In Eutopic Endometrial Stromal Cells Of Ovarian Endometriosis

Object:To investigate the relationship between the phosphorylation level of Cacy BP/SIP and the ubiquitin degradation level of β-catenin in eutopic endometrial stromal cells(ESCs)of Ovarian endometriosis(OEMs),and the effect of this dynamic process on the proliferation of ESCs.Methods:1.In vitro extract and culture ESCs from the patients with ovarian endometriosis,and identification.2.ESCs were treated with different concentrations of PMA(10n M,50 n M,and100 n M).The expression of total Cacy BP/SIP protein(t-Cacy BP),Cacy BP/SIP phosphorylation level(p-Cacy BP)and β-catenin level were detected by Western blot.3.ESCs treated with different concentrations of ionomycin(2.5um,5.0um and10.0um).The expression of total Cacy BP/SIP protein(t-Cacy BP),Cacy BP/SIP phosphorylation level(p-Cacy BP)and β-catenin expression were detected by Western blot.4.CCK-8 method was used to detect the proliferation ability of ESCs after treatment with different concentrations of ionomycin.5.Cell cycle was synchronized by thymidine block method,and using flow cytometry to measure the cell cycle.6.Extract total proteins from different periods,detecting the expression of total Cacy BP/SIP protein(t-Cacy BP),Cacy BP/SIP phosphorylation level(p-Cacy BP)andβ-catenin expression in ESCs with different phases.Results:1.ESCs from ovarian endometriosis patients were successfully isolated and cultured in vitro.2.After ESCs were treated with different concentrations of PMA for 2h,intracellular total Cacy BP/SIP(t-Cacy BP)expression and phosphorylation level of Cacy BP/SIP(p-Cacy BP)were significantly increased(P<0.05),and the differences have statistical significance.But the level of β-catenin decreased gradually(P<0.05),and the differences have statistical significance.3.After ESCs were treated with ionomycin at different concentrations for 30 min,intracellular total Cacy BP/SIP(t-Cacy BP)expression and phosphorylation level of Cacy BP/SIP(p-Cacy BP)were significantly increased(P<0.05),and the differences have statistical significance.But the level of β-catenin decreased gradually(P<0.05),and the differences have statistical significance.4.The results of cell proliferation test by CCK-8 method showed that the proliferation function of ESCs was significantly inhibited after treated with ionomycin at different concentrations for 24h(P<0.01),and the differences have statistical significance.5.After cell cycle synchronization by double thymidine block,most of the ESCs were blocked in the G1/S phase,and after 4 hours of withdrawal to cultivate,the cells entered the S phase.Most cells entered the G2 phase at 8-12 h,and then entered the G1 phase again at 16 h.6.There was no significant change in total Cacy BP/SIP(t-Cacy BP)at all time phases(compared with 0h,4h and 16 h,P>0.05),but the phosphorylation level of Cacy BP/SIP(p-Cacy BP)significantly increased at 8h and 12h(mainly in G2 phase cells)(compared with 0h,4h and 16 h,P<0.05),and the differences have statistical significance.At the same time,the expression of β-catenin decreased(compared with0 h,4h and 16 h,P<0.05),and the differences have statistical significance.Conclusion:In OEMs,the phosphorylation level of Cacy BP/SIP is negatively correlated with the level of β-catenin,and the increased phosphorylation level of Cacy BP /SIP can inhibit the proliferation of ESCs.