The Regulation Mechanism Of MicroRNA-223in Embryo Implantation

Part Ⅰ The expression of IGF-1R was regulated by miR-223in endometrium epithelial cellsObjective:The objective of this part is to investigate the target gene of miR-223in endometrium epithelial cells.Methods:TargetScan5.1was used to predict the target gene of miR-223. Lentivirus transfection was used to induce miR-223over-expression in RL95-2epithelial cell line. The expression of miR-223and predicted target genes were analysed by real-time PCR and/or Western Blot. Then, luciferase reporter assay was used to confirm the functional target of miR-223.Results:(1) IGF-1R、FOXO1、E2F1and IKKa was predicted as the target genes of miR-223;(2) The mRNA levels of IGF-1R、FOXO1、E2F1and IKKa were not changed when miR-223was over-expressed in RL95-2epithelial cells. However, the expression of IGF-1R protein was inhibited by over-expressed miR-223in RL95-2epithelial cells.(3) Luciferase reporter assay revealed that IGF-1R is a functional target of miR-223.Conclusion:Our data suggested that miR-223can bind to3’UTR of IGF-1R and regulate the expression of IGF-1R protein in endometrium epithelial cells. Part Ⅱ The expression of miR-223and IGF-1R in human endometriumObjective:To observe the expression of miR-223and IGF-1R in human endometrium of different periods.Methods:Real-time PCR was used to detect the level of miR-223and IGF-1R mRNA in mid-secretory and mid-proliferative endometria. Western Blot and immunohistochemical were used to explore the level and cell location of IGF-1R protein.Results:(1) The expression of miR-223mRNA in mid-secretory endometria was significantly lower than that in mid-proliferative endometria. However, the mRNA level of IGF-1R was similar between two groups.(2) The results of Western Blot revealed that the protein level of IGF-1R was significantly higher in mid-secretory endometria comparing to mid-proliferative endometria.(3) The results of immunohistochemical revealed that IGF-1R mainly located in the endometrium epithelial cells.Conclusion:Our data showed that negative correlation existed between the expression of miR-223and IGF-1R in human endometrium, which further confirmed our conclusion in cell experiments. Part Ⅲ Over-expression of miR-223inhibit embryonic implantationObjective:To investigate the effect of miR-223in embryonic implantationMethods:Using an in vitro model of endometrium-trophoblast interaction established with Bewo trophoblast and RL95-2cells, the endometrial receptivity was evaluated by implantation assay. In vivo implantation model was used to observe the effect of miR-223in embryo implantation.Results:(1) Over-expression of miR-223in RL95-2cells inhibited the attachment of trophoblast spheroids to EEC monolayer.(2) Treatment of mice uterine with miR-223agonist decreased the implantation rate of in vivo implantation model.Conclusion:The results of in vitro implantation model and in vivo mice model suggested that miR-223inhibited embryonic implantation via IGF-1R...