miR-181b-5p Modulates Cell Migratory Proteins, Tissue Inhibitor of Metalloproteinase 3, and Annexin A2 During In Vitro Decidualization in a Human Endometrial Stromal Cell Line.

Decidualization is essential for successful embryo implantation and is regulated by concerted actions of growth factors and hormones. More recently, microRNAs, small RNA molecules that regulate posttranscriptional gene expression, have been implicated to play a role in the decidualization process. Of these microRNAs, miR-181b-5p has been associated with decidualization but its precise role and targets are not well established. To address this gap in our knowledge, we assessed the expression of miR-181b-5p, and its target tissue inhibitor of metalloproteinase 3 (TIMP-3), during in vitro decidualization using the well-characterized human endometrial stromal cell line, t-HESC. miR-181b-5p expression was highest prior to decidualization and significantly decreased in response to decidualization stimulus. In contrast, TIMP-3 expression was absent prior to in vitro decidualization and increased during decidualization. Regulation of TIMP-3 expression by miR-181b-5p was confirmed in vitro by quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blot analysis, and 3' untranslated region reporter constructs. To identify unforeseen targets of miR-181b-5p during in vitro decidualization, t-HESC cells were transfected with pre- miR-181b-5p, and protein profiles were determined by 2-dimensional differential in-gel electrophoresis followed by matrix-assisted laser desorption-ionization time-of-flight/time-of-flight (MALDI TOF/TOF) tandem mass spectrometry. Of these proteins, several downregulated proteins associated with cell migration were identified including annexin A2, which we subsequently confirmed by qRT-PCR and Western blot analysis to be regulated by miR-181b-5p. In conclusion, miR-181b-5p is downregulated during the process of in vitro decidualization and may regulate the expression of proteins associated with cell migration including TIMP-3 and annexin A2.

miR-451 deficiency is associated with altered endometrial fibrinogen alpha chain expression and reduced endometriotic implant establishment in an experimental mouse model.

Endometriosis is defined as the growth of endometrial glandular and stromal components in ectopic locations and affects as many as 10% of all women of reproductive age. Despite its high prevalence, the pathogenesis of endometriosis remains poorly understood. MicroRNAs, small non-coding RNAs that post-transcriptionally regulate gene expression, are mis-expressed in endometriosis but a functional role in the disease pathogenesis remains uncertain. To examine the role of microRNA-451 (miR-451) in the initial development of endometriosis, we utilized a novel mouse model in which eutopic endometrial fragments used to induce endometriosis were deficient for miR-451. After induction of the disease, we evaluated the impact of this deficiency on implant development and survival. Loss of miR-451 expression resulted in a lower number of ectopic lesions established in vivo. Analysis of differential protein profiles between miR-451 deficient and wild-type endometrial fragments revealed that fibrinogen alpha polypeptide isoform 2 precursor was approximately 2-fold higher in the miR-451 null donor endometrial tissue and this elevated expression of the protein was associated with altered expression of the parent fibrinogen alpha chain mRNA and protein. As this polypeptide contains RGD amino acid "cell adhesion" motifs which could impact early establishment of lesion development, we examined and confirmed using a cyclic RGD peptide antagonist, that endometrial cell adhesion and endometriosis establishment could be respectively inhibited both in vitro and in vivo. Collectively, these results suggest that the reduced miR-451 eutopic endometrial expression does not enhance initial establishment of these fragments when displaced into the peritoneal cavity, that loss of eutopic endometrial miR-451 expression is associated with altered expression of fibrinogen alpha chain mRNA and protein, and that RGD cyclic peptide antagonists inhibit establishment of endometriosis development in an experimental mouse model suggesting that this approach may prove useful in the prevention of endometriosis establishment and survival.