| Background and Objective: 1.Fucosylation of proteins plays a significant role in determining the receptivity of the uterine endometrium to embryo.2.Fucosyltransferase IV(FUT4)is stage-specifically expressed in uterine endometrium of mammalians and considered as a marker of the endometrial receptivity.3.micro RNA is a kind of endogenous non-coding small molecule RNA,and miR-200 c is a member of miR-200 family which involved in human reproduction.However,the relationship between miR-200 c and FUT4,and the role of which in embryo-endometrium adhesion remains unclear.4.In this study,we focus on the effection of miR-200 c on embryo adhesion/implantation and its underlying glycobiological mechanism,which further explain the important regulation of miR-200 c on mammalian embryo implantation.Method: 1.We performed Real-time PCR,ELISA and western blot experiments to determine the expression level of miR-200 c and FUT4 in serum of healthy non-pregnancy,infertility,pregnancy and abortion patients.2.CCK-8 assay and colony formation assay were performed to assess the effect of miR-200 c on cell proliferation.We next investigated the potential role of miR-200 c in modulating the ability of uterine epithelium cells to receptivity formulation.3.We compared sequences between miR-200 c and FUT4 by searching over 10 databases,and performed Dual-Luciferase Reporter Assay to find out whether FUT4 is the target gene of miR-200 c.4.For further research of the role that miR-200 c plays in regulation of implantation,we studied Wnt/β-catenin signaling pathway.Results: 1.Real-time PCR,ELISA and western blot data showed that expression of miR-200 c increased in infertility patients serum compared with healthy non-pregnancy women.Whereas,miR-200 c level increased in abortion patients’ serum compared with pregnancy women.The expression of FUT4 decreased in infertility patients serum compared with healthy non-pregnancy women,and FUT4 level decreased in abortion patients’ serum compared with pregnancy women.2.miR-200 c inhibit proliferation and receptivity of uterine epithelium cell in vitro.CCK-8 assay and colony formation assay were performed to assess the effect of miR-200 c on cell proliferation.RL95-2 cells transfected with miR-200 c mimic showed a lower proliferation rate and fewer numbers of colonies than control cells.In contrast,RL95-2 cells expressing lower miR-200 c exhibited higher proliferation rate and greater number of colonies than control cells.The embryo implantation model in vitro also showed RL95-2 cells overexpressing miR-200 c exhibited significantly reduced rate of adhesion,compared to control cells,whereas RL95-2 cells decreased miR-200 c adhesion fewer,relative to control cells.3.FUT4 is a target gene of miR-200 c.The results showed that the luciferase activity of FUT4 significantly decreased after co-transfection of WT luciferase reporter constructs and miR-200 c mimics,whereas we have little change in activity when co-transfected MUT luciferase reporter constructs and miR-200 c mimics in the two kinds of uterine epithelium cells.Real-time PCR and Western blot were performed.The data showed that miR-200 c mimics could significantly down regulated FUT4 expression while Anti-miR-200 c induced FUT4 up regulation.These results were confirmed after co-transfection of miR-200 c mimics and FUT4 c DNA,and also after co-transfection of Anti-miR-200 c and FUT4 si RNA,which significantly restored or reduced FUT4 protein level,respectively.Immunofluorescent staining of FUT4 was also observed the similar changes.4.miR-200 c regulated functions of endometrial cells by inactivating Wnt/β-catenin signaling pathway.p-GSK3β and β-catenin were inactivated after transfected with miR-200 c mimic,whereas restored FUT4 expression by induced in FUT4 c DNA could reactivated Wnt/β-catenin signal pathway.And also,anti-miR-200 c promoted p-GSK3β and β-catenin expression,silenced FUT4 by specific si RNA could inactivate Wnt/β-catenin signal pathway in RL95-2 cells.Besides,DKK(inhibitor of Wnt/β-catenin signal pathway)inactivated Wnt/β-catenin signal pathway.Conclusion: 1.miR-200 c can be tested in serum and its expression has a negative correlation to FUT4.2.Down-regulating miR-200 c enhances human endometrial RL95-2 cells adhesion to human embryonic JAR cells.3.miR-200 c targets on FUT4.4.miR-200 c decreases endometrial receptivity by down-regulation of FUT4 which mediated by Wnt/β-catenin signaling pathway. |
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