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.
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References
Bartel DP. MicroRNAs: genomics, biogenesis, mechanism and function. Cell. 2004;116(2):281–297.
Vasudevan S, Tony Y, Steitz JA. Switching from repression to activation: microRNAs can up-regulate translation. Science. 2007;318(5858):1931–1934.
Lee S, Vasudevan S. Post-transcriptional stimulation of gene expression by microRNAs. Adv Exp Med Biol. 2013;768(1):97–126.
Brümmer A, Hausser J. MicroRNA binding sites in the coding regions of mRNAs: extending the repertoire of post-transcriptional gene regulation. Bioessays. 2014;36(1):617–626.
Nagaraja AK, Andreu-Vieyra C, Franco HL, et al. Deletion of dicer in the somatic cells of the female reproductive tract causes sterility. Mol Endocrinol. 2008;22(10):2336–2352.
Hong X, Luense LJ, McGinnis LK, Nothnick WB, Christenson LK. Dicer1 is essential for female fertility and normal development of the female reproductive system. Endocrinology. 2008;149(12):6207–6212.
Kim BM, Woo J, Kanellopoulou C, Shivdasani RA. Regulation of mouse stomach development and Barx1 expression by specific microRNAs. Development. 2011;138(6):1081–1086.
Nothnick WB, Healy C. Estrogen induces distinct patterns of microRNA expression within the mouse uterus. Reprod Sci. 2010;17(11):987–994.
Lessey BA. Fine tuning of endometrial function by estrogen and progesterone through microRNAs. Biol Reprod. 2010;82(4):653–655.
Kuokkanen S, Chen B, Ojalvo L, Benard L, Santoro N, Pollard JW. Genomic profiling of microRNAs and messenger RNAs reveals hormonal regulation in microRNA expression in human endometrium. Biol Reprod. 2010;82(4):791–801.
Estella C, Herrer I, Moreno-Moya JM, et al. miRNA signature and Dicer requirement during human endometrial stromal decidualization in vitro. PLoS One. 2012;7(7):e41080.
Liu G, Min H, Yue S, Yue S, Chen CZ. Pre-miRNA loop nucleotides control the distinct activities of mir-181a-1 and mir181c in early T cell development. PLoS One. 2008;3++(10):e3592.
Ji J, Yamashita T, Wang XW. Wnt/beta-catenin signaling activates microRNA-181 expression in hepatocellular carcinoma. Cell Biosci. 2011;1(1):4.
Agarwal V, Bell GW, Nam JW, Bartel DP. Predicting effective microRNA target sites in mammalian mRNAs. Elife. 2015;12:4.
Miranda KC, Huynh T, Tay Y, et al. A pattern-based method for the identification of microRNA binding sites and their corresponding heteroduplexes. Cell. 2006;126(6):1203–1217.
Christian M, Mak I, White JO, Brosens JJ. Mechanisms of decidualization. Reprod Biomed Online. 2002;4(suppl 3):24–30.
Ramathal CY, Bagchi IC, Taylor RN, Bagchi MK. Endometrial decidualization: of mice and men. Semin Reprod Med. 2010;28(1):17–26.
Pawar S, Hantak AM, Bagchi IC, Bagchi MK. Minireview: steroid-regulated paracrine mechanisms controlling implantation. Mol Endocrinol. 2014;28(9):1408–1422.
Reponen P, Leivo I, Sahlberg C, et al. 92-kDa type IV collagenase and TIMP-3, but not 72-kDa type IV collagenase or TIMP-1 or TIMP-2, are highly expressed during mouse embryo implantation. Dev Dyn. 1995;202(4):388–396.
Alexander CM, Hansell EJ, Behrendtsen O, et al. Expression and function of matrix metalloproteinases and their inhibitors at the maternal-embryonic boundary during mouse embryo implantation. Development. 1996;122(6):1723–1736.
Leco KJ, Edwards DR, Schultz GA. Tissue inhibitor of metalloproteinases-3 is the major metalloproteinase inhibitor in the decidualizing murine uterus. Mol Reprod Dev. 1996;45(4):458–465.
Bany BM, Schultz GA. Tissue inhibitor of matrix metalloproteinase-3 expression in the mouse uterus during implantation and artificially induced decidualization. Mol Reprod Dev. 2001;59(2):159–167.
Higuchi T, Kanzaki H, Nakayama H, et al. Induction of tissue inhibitor of metalloproteinase 3 gene expression during in vitro decidualization of human endometrial stromal cells. Endocrinology. 1995;136(11):4973–4981.
Huang HY, Wen Y, Irwin JC, Kruessel JS, Soong YK, Polan ML. Cytokine-mediated regulation of 92-kilodalton type IV collagenase, tissue inhibitor or metalloproteinase-1 (TIMP-1), and TIMP-3 messenger ribonucleic acid expression in human endometrial stromal cells. J Clin Endocrinol Metab. 1998;83(5):1721–1729.
Gao F, Chen XL, Wei P, Gao HJ, Liu YX. Expression of matrix metalloproteinase-2, tissue inhibitors of metalloproteinase-1, -3 at the implantation site of rhesus monkey during the early stage of pregnancy. Endocrine. 2001;16(1):47–54.
Krikun G, Mor G, Alvero A, et al. A novel immortalized human endometrial stromal cell line with normal progestational response. Endocrinology. 2004;145(5):2291–2296.
Pabona JM, Zeng Z, Simmen FA, Simmen RC. Functional differentiation of uterine stromal cells involves cross-regulation between bone morphogenetic protein 2 and Kruppel-like factor (KLF) family members KLF9 and KLF13. Endocrinology. 2010;151(7):3396–3406.
Nothnick WB, Graham A, Holbert J, Weiss MJ. miR-451 deficiency is associated with altered endometrial fibrinogen alpha chain expression and reduced endometriotic implant establishment in an experimental mouse model. PLoS One. 2014;9(6):e100336.
Wang B, Hsu SH, Majumder S, et al. TGFbeta-mediated upregulation of hepatic miR-181b promotes hepatocarcinogenesis by targeting TIMP3. Oncogene. 2010;29(12):1787–1797.
Lv Y, Gao S, Zhang Y, Wang L, Chen X, Wang Y. miRNA and target gene expression in menstrual endometria and early pregnancy decidua. Eur J Obstet Gynecol Reprod Biol. 2016;197:27–30.
Liu JL, Su RW, Yang ZM. Differential expression profiles of mRNAs, miRNAs and proteins during embryo implantation. Front Biosci (Schol Ed). 2011;3:1511–1519.
Galliano D, Pellicer A. MicroRNA and implantation. Fertil Steril. 2014;101(6):1531–1544.
Siristatidis C, Vogiatzi P, Brachnis N, et al. Review: MicroRNAs in assisted reproduction and their potential role in IVF failure. In Vivo. 2015;29(2):169–175.
Chu B, Zhong L, Dou S, et al. miRNA-181 regulates embryo implantation in mice through targeting leukemia inhibitory factor. J Mol Cell Biol. 2015;7(1):12–22.
Lu Y, Roy S, Nuovo G, et al. Anti-microRNA-222 (anti-miR-222) and -181B suppress growth of tamoxifen-resistant xenografts in mouse by targeting TIMP3 protein and modulating mitogenic signal. J Biol Chem. 2011;286(49):42292–42302.
Guo JX, Tao QS, Lou PR, Chen XC, Chen J, Yuan GB. miR-181b as a potential molecular target for anticancer therapy of gastric neoplasms. Asian Pac J Cancer Prev. 2012;13(5):2263–2267.
Zhou Q, Zheng X, Chen L, et al. Smad2/3/4 pathway contributes to TGF-β-induced MiRNA-181b expression to promote gastric cancer metastasis by targeting TIMP3. Cell Physiol Biochem. 2016;39(2):453–466.
Domínguez F, Garrido-Gómez T, López JA, et al. Proteomic analysis of the human receptive versus non-receptive endometrium using differential in-gel electrophoresis and MALDI-MS unveils stathmin 1 and annexin A2 as differentially regulated. Hum Reprod. 2009;24(10):2607–2617.
Garrido-Gómez T, Dominguez F, Quiñonero A, et al. Annexin A2 is critical for embryo adhesiveness to the human endometrium by RhoA activation through F-actin regulation. FASEB J. 2012;26(9):3715–3727.
Wang B, Ye TM, Lee KF, et al. Annexin A2 acts as an adhesion molecule on the endometrial epithelium during implantation in mice. PLoS One. 2015;10(10):e0139506.
Hayes MJ, Shao D, Bailly M, Moss SE. Regulation of actin dynamics by annexin 2. EMBO J. 2006;25(9):1816–1826.
Ihnatovych I, Livak M, Reed J, de Lenerolle P, Strakova Z. Manipulating actin dynamics affects human in vitro decidualization. Biol Reprod. 2009;81(1):222–230.
Sheng M. PDZs and receptor/channel clustering: rounding up the latest suspects. Neuron. 1996;17(4):575–578.
Rosenbaum J. Cytoskeleton: functions for tubulin modifications at last. Curr Biol. 2000;10++(21):R801–R803.
Ding Z, Lambrechts A, Parepally M, Roy P. Silencing profilin-1 inhibits endothelial cell proliferation, migration and cord morphogenesis. J Cell Sci. 2006;119(pt 9):4127–4137.
Kullmann JA, Neumeyer A, Gurniak CB, Friauf E, Witke W, Rust MB. Profilin1 is required for glial cell adhesion and radial migration of cerebellar granule neurons. EMBO Rep. 2011;13(1):75–82.
Das AM, Bolkestein M, van der Klok T, et al. Tissue inhibitor of metalloproteinase-3 (TIMP3) expression decreases during melanoma progression and inhibits melanoma cell migration. Eur J Cancer. 2016;66:34–46.
Xu C, Hou Z, Zhan P, et al. EZH2 regulates cancer cell migration through repressing TIMP-3 in non-small cell lung cancer. Med Oncol. 2013;30(4):713.
Weimar CH, Macklon NS, Post Uiterweer ED, Brosens JJ, Gellersen B. The motile and invasive capacity of human endometrial stromal cells: implications for normal and impaired reproductive function. Hum Reprod Update. 2013;19(5):542–557.
Menkhorst EM, Lane N, Winship AL, et al. Decidual-secreted factors alter invasive trophoblast membrane and secreted proteins implying a role for decidual cell regulation of placentation. PLoS One. 2012;7(2):e31418.
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Graham, A., Holbert, J. & Nothnick, W.B. 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. Reprod. Sci. 24, 1264–1274 (2017). https://doi.org/10.1177/1933719116682877
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DOI: https://doi.org/10.1177/1933719116682877