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

Abstract

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.

Construction of a MicroRNA-mRNA Network Underlying Decidualized Endometriotic Cyst Stromal Cells Using Bioinformatics Analysis

“…In our study, 9 miRNAs were screened out (miRNA-378a-3p, miRNA-7-5p, miRNA-55-5p, miRNA-18a-5p, and miRNA-18b-5p were low expressed in decidual ECSCs, and miRNA-30d-5p, miRNA-30b-5p, miRNA-181c-5p, and miRNA-766-3p were highly expressed in decidual ECSCs). According to previous research reports, downregulation of mir-378a-3p [ 35 ], miR-181b-5p [ 36 ], and miR-155 [ 37 ] is beneficial to the formation of decidualization. This is in agreement with our results.…”

Analysis of Reciprocally Dysregulated miRNAs in Eutopic Endometrium Is a Promising Approach for Low Invasive Diagnostics of Adenomyosis

“…For instance, miR-191 was found to be over-expressed in endometriomas tissue and to be a key regulator in the proliferation and the invasive properties of endometriosis cells (CRL7566) in vitro [ 32 ]. MiR-181b is involved in the regulation of the endometrial stromal cells’ migratory capacity [ 33 ]. The effect of miR-10b down-regulation in the endometrial cells may be realized via Syndecan-1, resulting in increased invasiveness [ 34 ].…”

TGFβ superfamily signaling and uterine decidualization

“…For example, the levels of miR-542-3p are lower in decidualizing versus normal human ESCs, and overexpression of miR-542-3p inhibits the expression of IGFBP1 , PRL, and WNT4 , suggesting an inhibitory role of miR-542-3p in decidualization [ 94 ]. It has also been reported that miR-181b-5p regulates the expression of cell migration associated proteins during decidualization [ 95 ]. Although TGFβ signaling regulates miRNA biosynthesis/expression [ 96 – 98 ], little is known about interactions between TGFβ signaling and miRNAs in the regulation of decidualization.…”