MicroRNA223-3p Regulates Embryo Implantation Via Suppressing LIF Expression And Pinopode Formation

Part ?. The expression pattern of miRNA223-3p and LIF in human endometriumObjective:To investigate the expression pattern of miRNA-223-3p (miRNA223) and LIF in the human endometrium of proliferative and mid-secretory phase.Methods:Real-time PCR was performed to examine the expression level of miRNA223 and LIF mRNA. The immunohistochemistry (IHC) was used to evaluate the expression and intra-cellular location of LIF protein.Results:The expression level of miRNA223 was higher in the proliferative endometrium compared with the mid-secretory endometrium. The expression of mRNA and protein of LIF were lower in the proliferative endometrium compared with the mid-secretory endometrium. LIF was strongly expressed in luminal and glandular epithelium cells; it was mildly expressed by the stromal cells.Conclusions:The menstrual cycle-dependent pattern of miRNA223 and LIF suggests that they are probably associated with the initiation of implantation window. There is a minus association between miRNA223 and LIF, indicating that miRNA223 might regulate LIF during embryo implantation. However, further study was needed to confirm these hypotheses.Part ?. MicroRNA-223-3p inhibits embryo implantation via down-regulating LIF expression and pinopode formation in miceObjective:To compare the expression of LIF and miRNA223 in the endometrium of pregnant and non-pregnant mice, and to investigate whether miRNA223 inhibits embryo implantation via suppressing the expression of LIF and pinopode.Methods:RT-PCR was used for the examination of the expression of miRNA223 and LIF. Western Blot and IHC were performed to evaluate the protein of LIF. The miRNA223-Agomir was injected into the left side of uterus horn of pregnant mice on Day 3 morning, the expression level of miRNA223 and LIF mRNA on Day 4 afternoon were examined by RT-PCR. Transparent-electronic scope was used to evaluate the formation of pinopode. Some mice were sacrificed on Day 9 to count the number of implanted embryos.Results:The expression of miRNA223 was higher in the non-pregnant mice compared with the pregnant mice; in the contrast, LIF was lower in the non-pregnant mice than the pregnant ones. In the pregnant mice, the expression of miRNA223 was elevated, while the expression of LIF protein was reduced in the side of intervention with miRNA223-Agomir, compared with the side without intervention. Transparent-electronic scope showed that the formation of pinopode was affected by miRNA223-Agomir. The number of implanted embryos was also decreased after administration with miRNA223-Agomir.Conclusions:miRNA223 has a negative effect on embryo implantation, which is probably via suppressing the expression of LIF and pinopode. Further study addressing whether miRNA223 directly interacts with LIF should be performed.Part ?. microRNA-223-3p directly interacts with LIF and compromises the cytoskeleton of endometrial epithelium in vitro.Objective:To address whether miRNA223 directly influences LIF, and to investigate the effect of miRNA223 on cytoskeleton of endometrial luminal epithelial cells.Methods:The luciferase assay was used to examine the functional relation between miRNA223 and LIF. The endometrial luminal epithelial cell lines (HES and RL95-2) were transfected with Pre-miRNA223 clone. Real-Time PCR and Western Blot were used to evaluate whether the transfection succeeded. Immunologic cyto-chemistry (ICC) was used to evaluate the cytoskeleton of HES and RL95-2 cells, using Amanita Phalloides (AP) and ?-Tublin (BT) as parameters.Results:Pre-miRNA223 clone inhibits LIF via binding its 3'UTR. The miRNA223 expression was elevated while the LIF expression was decreased after intervention with Pre-miRNA223 clone. The AP and BT expression were both reduced after administration with Pre-miRNA223 clone in HES cells, while only the AP expression was reduced in RL95-2 cells.Conclusions:miRNA223 directly suppresses LIF, and thus compromises the cytoskeleton of endometrial luminal epithelial cells, which is probably associated with aberrant formation of pinopode.