Galectin-1调节子宫内膜容受性的机制研究

Effect of Galectin-1 on the function of endometrial epithelial cells with different receptivity

目的:探讨Galectin-1对子宫内膜容受性的影响以及在胚泡着床过程中所起的作用。方法:采用实时荧光定量PCR法、Western blotting及细胞免疫荧光法检测Galectin-1在高容受性子宫内膜细胞RL-95-2和低容受性子宫内膜细胞HEC-1-A中的表达及定位情况,通过Transwell实验和划痕实验检测两种细胞的侵袭和迁移能力。用JAR细胞模拟胚胎,测JAR细胞在RL-95-2和HEC-1-A上的黏附能力。将RL-95-2与HEC-1-A分别转染Galectin-1 siRNA和Galectin-1过表达载体后,Western blotting检测细胞中Galectin-1的表达水平、上皮间质转化(epithelial-mesenchymal transition,EMT)相关蛋白(E-cadherin、N-cadherin和Vimentin蛋白)及WNT/β-catenin信号通路相关蛋白(β-catenin、WNT4a、WNT5a和WNT7a蛋白)的表达,同时检测细胞的侵袭、迁移情况。将转染后的细胞分别加入WNT通路抑制剂DKK1及激活剂氯化锂(LiCl),检测EMT和WNT相关蛋白表达水平及细胞的侵袭、迁移情况。结果:①RT-PCR和Western blotting结果显示,Galectin-1在RL-95-2中的表达量明显高于其在HEC-1-A中的表达(P=0.020,P=0.030);细胞免疫荧光实验显示,Galectin-1主要表达于RL-95-2细胞质,而在HEC-1-A细胞中,Galectin-1主要表达于细胞核。②细胞黏附实验结果表明,JAR细胞在RL-95-2细胞的黏附率明显高于HEC-1-A细胞(P=0.010)。③Galectin-1过表达后,HEC-1-A细胞中E-cadherin蛋白表达量降低(P=0.001),N-cadherin和Vimentin蛋白表达量升高(P=0.003,P=0.023);WNT相关蛋白β-catenin、WNT4a和WNT5a、WNT7a蛋白表达量升高(P=0.025,P=0.004,P=0.005,P=0.001),细胞的迁移能力、侵袭能力明显增强(P=0.022,P=0.003)。④DKK1处理过表达Galectin-1的HEC-1-A细胞后,与DKK1处理HEC-1-A细胞相比,E-cadherin蛋白表达量降低(P=0.003),N-cadherin和Vimentin蛋白表达量升高(P=0.015,P=0.033),细胞的迁移能力以及侵袭能力均明显增加(P=0.030,P=0.040)。⑤RL-95-2细胞下调Galectin-1的表达后,E-cadherin蛋白表达量升高(P=0.004),N-cadherin和Vimentin蛋白表达量明显降低(P=0.030,P=0.023),β-catenin、WNT4a、WNT5a、WNT7a蛋白表达量降低(P=0.001,P=0.005,P=0.023,P=0.020)。⑥LiCl处理下调Galectin-1表达的RL-95-2细胞后,与LiCl处理RL-95-2细胞相比,E-cadherin蛋白表达量增加,N-cadherin蛋白和Vimentin蛋白表达量降低(P=0.012,P=0.035,P=0.020);细胞迁移率、侵袭数目降低(P=0.040,P=0.020)。结论:与低容受性子宫内膜细胞相比,高容受性的子宫内膜细胞中Galectin-1的表达水平增加;Galectin-1可能通过WNT/β-catenin信号通路调控EMT相关蛋白的表达,从而影响胚胎的着床。

Objective To observe the expression of Galectin-1 in human ectopic endometrial cells with different receptivity and its role in the process of embryo implantation.Methods The expression and localization of Galectin-1 in high tolerance endometrial cell RL-95-2 and low tolerance endometrial cell HEC-1-A were detected by real-time quantitative PCR(RT-qPCR),Western blotting and immunofluorescence assay.The invasion and migration of the two kinds of cells were detected by Transwell assay and scratch assay.JAR cells were used to mimic embryos and to detect the migration rate.The expression levels of Galectin-1,epithelial-mesenchymal transition(EMT)-related proteins and WNT/β-catenin signaling pathway-related proteins were analyzed by transfecting Galection-1 siRNA and Galection-1 overexpression vector in the RL-95-2 cells and HEC-1-A cells,respectively.Then the transfected endometrial cells were added with WNT pathway inhibitor DKK1 and activator LiCl respectively,and the changes of EMT-related proteins and the invasion,migration of cells were examined.Results 1)RT-qPCR and Western blotting results showed that Galectin-1 was significantly higher in RL-95-2 than in HEC-1-A(P=0.020,P=0.030);immunofluorescence experiments showed that Galectin-1 was mainly expressed in the cytoplasm of RL-95-2,while in HEC-1-A cells,Galectin-1 was mainly expressed in the nucleus.2)The results of cell adhesion assay showed that the adhesion rate of JAR cells to RL-95-2 cells was significantly higher than that to HEC-1-A cells(P=0.010).3)After Galectin-1 overexpression,the expression of E-cadherin protein was decreased in the HEC-1-A cells(P=0.001),and the expressions of N-cadherin and Vimentin were increased(P=0.003,P=0.023);the exprssioins ofβ-catenin,WNT4a,WNT5a,and WNT7a were increased(P=0.025,P=0.004,P=0.005,P=0.001),and the migration ability and invasion ability of HEC-1-A cells were significantly enhanced(P=0.022,P=0.003).4)After DKK1 treatment of HEC-1-A cells overexpressing Galectin-1,E-cadherin protein expression was decreased(P=0.003),N-cadherin and Vimentin protein expressions were increased(P=0.015,P=0.033),and the migratory ability as well as invasive ability of HEC-1-A cells were significantly increased(P=0.030,P=0.040).5)After down-regulation of Galectin-1 expression in RL-95-2 cells,E-cadherin protein expression was increased(P=0.004),N-cadherin and Vimentin protein expressions were significantly decreased(P=0.030,P=0.023),andβ-catenin,WNT4a,WNT5a,and WNT7a protein expressions were reduced(P=0.001,P=0.005,P=0.023,P=0.020).6)After LiCl treatment of RL-95-2 cells with down-regulated Galectin-1 expression,E-cadherin protein expression was increased,and N-cadherin protein and Vimentin protein expressions were decreased compared with LiCl-treated RL-95-2 cells(P=0.012,P=0.035,P=0.020);RL-95-2 cells migration rate,and invasion number were decreased(P=0.040,P=0.020).Conclusion The expression level of Galectin-1 was increased in highly receptivity endometrium compared with low receptivity endometrium.Galectin-1 may regulate the expression of EMT-related proteins through WNT/β-catenin signaling pathway,thus affecting embryo implantation.