{"paper_id":"cd6e6510-71d6-4d79-9dfa-e701942008f1","body_text":"R E S E A R C H Open Access\nMicroRNA-181a is involved in the regulation of\nhuman endometrial stromal cell decidualization\nby inhibiting Krüppel-like factor 12\nQun Zhang 1† , Hui Zhang 2† , Yue Jiang 1, Bai Xue 2, Zhenyu Diao 1, Lijun Ding 1, Xin Zhen 1, Haixiang Sun 1,\nGuijun Yan 1* and Yali Hu 1*\nAbstract\nBackground: The transformation of endometrium into decidua is essential for normal implantation of the blastocyst.\nHowever, the post-transcriptional regulation and the miRNAs involved in decidualization remain poorly understood.\nHere, we examined microRNA-181a (miR-181a) expression in decidualized human endometrial stromal cell (hESC). In\naddition, we investigated the functional effect of miR-181a on hESC decidualization in vitro.\nMethods: Quantitative real-time PCR (qRT-PCR) was used to de tect the profile of miR-181a in decidualized hESC.\nqRT-PCR, enzyme-linked fluorescent assay, and immunofluoresc e n c ea s s a yw e r ep e r f o r m e dt oi n v e s t i g a t ed e c i d u a l i z a t i o n\nmarker genes’ expression after enhancing or inhibition of miR-181a expression in hESC. Luciferase reporter assay, western\nblotting, qRT-PCR, and immunofluorescence assay were carried out to identify the relationship between miR-181a and\nKrüppel-like factor 12 (KLF12).\nResults: miR-181a expression levels increased dramatically in hESC treated with 8-Br-cAMP and MPA. Increased miR-181a\nexpression promoted hESC decidualization-related gene e xpression and morphological transformation; conversely,\ninhibition of miR-181a expression compromised hESC decidualization in vitro . Further analysis confirmed that\nmiR-181a interacted with the 3 ′ untranslated region of the transcription factor KLF12 and down-regulated KLF12 at\nthe transcriptional and translational l evels. KLF12 overexpression abolishe d miR-181a-induced decidualization.\nConclusions: Our findings suggest that miR-181a plays a functionally important role in human endometrial\ns t r o m a lc e l ld e c i d u a l i z a t i o nin vitro by inhibiting KLF12.\nKeywords: MicroRNA-181a, KLF12, Human endometr ial stromal cell, Decidualization\nBackground\nDecidualization of the endometrial stroma is a precondi-\ntion for the successful establishment of pregnancy. In\nhumans, this process is initiated in the mid-secretory\nphase of the menstrual cycle and is triggered by ovarian\nsex steroid hormones independent of pregnancy [1,2].\nThe decidual reaction consists of a dramatic morpho-\nlogical and biochemical transformation of the endomet-\nrial stroma in which the stromal fibroblasts differentiate\nto become rounded, relatively large epithelioid-like or\npolygonal, secretory decidual cells [3]. Human decidual\ncells produce specific molecules, such as regulatory fac-\ntors (prolactin (PRL) and insulin-like growth factor\nbinding protein-1 (IGFBP-1)), inflammatory mediators\n(IL-1, IL-6, IL-8, and TNF- α), and specific extracellular\nmatrix proteins (laminin, type IV collagen, and fibronec-\ntin) [4-7]. PRL and IGFBP-1 levels are generally used as\nbiochemical decidualization markers of progestin-induced\nhuman endometrial stromal cell (hESC) differentiation. A\nnumber of transcription factors and autocrine/paracrine\nfactors have been identified that cooperatively control the\ndecidualization process. However, little information is\navailable regarding the post-transcriptional regulation of\nthis process.\n* Correspondence: yanguijun55@163.com; glyyhuyali@hotmail.com\n† Equal contributors\n1Reproductive Medicine Center, Department of Obstetrics and Gynecology,\nNanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing\n210008, Jiangsu, China\nFull list of author information is available at the end of the article\n© 2015 Zhang et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative\nCommons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and\nreproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain\nDedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,\nunless otherwise stated.\nZhang et al. Reproductive Biology and Endocrinology  (2015) 13:23 \nDOI 10.1186/s12958-015-0019-y\n\nMicroRNAs (miRNAs) have emerged as key post-\ntranscriptional regulators. An estimated 30 – 50% of\nprotein-coding genes serve as potential miRNA targets.\nmiRNAs regulate and influence a variety of cellular ac-\ntivities, including cell growth, differentiation, apoptosis,\nand metabolism [8]. miRNAs are small (approximately\n20– 22 nt), noncoding RNAs that generally base-pair\nwithin the 3 ′ untranslated region (3 ′UTR) of target\nmRNAs, causing translational inhibition and/or mRNA\ndegradation [9]. Recently, the conditional inactivation of\nDicer has provided evidence for the pivotal functions of\nmiRNAs in ovarian as well as oviductal and uterine stro-\nmal cell development [10]. Dicer expression increases\nand is a requirement during human endometrial stromal\ndecidualization in vitro [11]. The aberrant expression of\nsome miRNAs has been correlated with various endomet-\nrial diseases, such as endometriosis, repeated implantation\nfailure (RIF), and endometrial cancer [12-14].\nMicroRNA-181a (miR-181a), which belongs to the\nmiR-181 family, is a key modulator of cellular differenti-\nation. Based on microRNA microarray analysis, L. Su\net al. found high miR-181a/c expression on day 15 of\ngestation, followed by decreased expression on gesta-\ntional days 26 and 50 in the porcine endometrium dur-\ning pregnancy [15]. Various potential miR-181 family\ntargets, such as ETS1, CREB1/3, Esr1, and PGR, are in-\nvolved during differentiation and decidualization events\n[16-18]. The Krüppel-like factor (KLF) family members\nare revealed to play critical roles in regulating the\nprocess of embryo implantation, such as KLF9 and\nKLF13 [19,20]. KLF12, another member of KLF family,\nbinds to the CAGTGGG sequence within target gene\npromoter regions and represses target gene expression\nthrough an N-terminal PVDLS sequence (Pro-Xaa-Asp-\nLeu-Ser) that promotes a physical interaction with the\nco-repressor CtBPs [21]. We previously demonstrated\nthat KLF12, suppressed by 8-Br-cAMP and MPA, nega-\ntively regulates hESC decidualization by inhibiting PRL\nand IGFBP-1 expression [22]. In this study, we demon-\nstrated that miR-181a was involved in the regulation of\nhESC decidualization by suppressing KLF12, whereas\nKLF12 overexpression inhibited miR-181a-mediated in-\ncreases in decidualization-related gene expression and\nthe morphological transformation of hESC, indicating\nthat miR-181a may play an important role in human\nendometrial decidualization.\nMethods\nIsolation and in vitro decidualization of hESC\nThis study was approved by the Institutional Review\nBoard of the Drum Tower Hospital of Nanjing University\non December 5, 2013 (2013-081-01). This study was con-\nducted in the Drum Tower Hospital from February 2014\nto September 2014. Patient consent was received before\nbiopsy. hESC were isolated from the mid-secretory phase\nof endometrial tissue of women with a normal menstrual\ncycle by endometrial biopsy. hESC were isolated and cul-\ntured as previously described [23].\nTo induce decidualization, hESC were cultured in\nphenol red-free DMEM/F12 medium (HyClone, Thermo\nScientific, South Logan, UT, USA) containing 2.5%\ncharcoal/dextran-treated fetal bovine serum (FBS, HyClone,\nThermo Scientific, South Logan, UT, USA), 100 IU/ml\npenicillin, and 100 μg/ml of streptomycin supple-\nmented with 0.5 mM 8-Br-cAMP and 1 μMM P A\n(Sigma, St. Louis, MO, USA) for 3 days, 6 days, or\n9 days. Differentiation was assessed by examination of\ncell morphology under phase contrast microscopy at\nvarious times during the treatment and also by meas-\nuring the expression of decidu alization-specific marker\ngene, namely PRL.\nConstruction of adenovirus\nTo overexpress KLF12 and miR-181a in hESC, adenovirus\nvectors harboring the full-length KLF12 (Ad-KLF12) and\nprecursor miR-181a (Ad-miR-181a) were generated using\nthe AdMax (Microbix Biosystems, Inc., Toronto, Canada)\nand pSilencer ™ adeno 1.0-CMV (Ambion, Austin, TX,\nUSA) systems as previously described [22,24]. The\nprimers for full-length KLF 12 amplification were: 5 ′-T\nCTCGAATTCAATGAATATCCATATGAAGAG-3′ and\n5′-TATAGGATCCTCACACCAACATATGCCTCC-3′;\nfor precursor miR-181a amplification were: 5 ′-CGCGC\nTCGAGATACAATGTGATGTGGAGGTT-3′ and 5 ′-G\nCGCGATATCGGCCACAGTTGCATTCATTGT-3′.A n\nadenovirus bearing LacZ (Ad-LacZ) was obtained from\nClontech (Palo Alto, CA, USA) and used as the control of\nadenovirus infection experiments. The viruses were\npackaged and amplified in HEK293A cells and purified\nusing CsCl banding followed by dialysis against 10 mM\nTris-buffered saline with 10% glycerol. The viral titer\nwas determined using HEK293A cells and the Adeno-X\nRapid Titer kit (Clontech). hESC were infected with\nAd-miR-181a at the 100 MOI and/or with Ad-KLF12\nat the 20 MOI.\nmiRNA inhibitor transfection\nmiR-181a inhibitor (2 ′-O-methyl modified oligonu-\ncleotides: 5 ′-mAmCmUmCmAmCmCmGmAmCmAmG\nmCmGmUmUmGmAmAmUmGmUmU-3 ′)o rm i R N A\ninhibitor negative control (miRNA inhibitor control)\nwere synthesized by Ribobio (Guangzhou, China). miRNA\ninhibitor control shares no homologous region with\nthe human genome sequences. For loss of function\nexperiments, hESC were transfected with 100 nM of\nmiR-181a inhibitor or of miRNA inhibitor control\nusing Lipofectamine 2000 (Life Technologies, New\nYork, USA).\nZhang et al. Reproductive Biology and Endocrinology  (2015) 13:23 Page 2 of 9\n\nRNA isolation and quantitative real-time PCR (qRT-PCR)\nTotal RNA was extracted from hESC using Trizol reagent\n(Invitrogen, Carlsbad, CA, USA). Reverse transcription\nwas performed using random primers or specific miRNA\nstem-loop primers, and qRT-PCR was performed on a\nMyiQ Single-Color Real-Time PCR Detection System\n(BIO-RAD, Hercules, CA, USA). To detect miR-181a ex-\npression, we used the following primers: forward, 5 ′-AC\nACTCCAGCTGGGAACATTCAACGCTGTCG-3 ′;r e -\nverse, 5 ′-GGTGTCGTGGAGTCGGCAATTCAGTTG\nAG-3′. The small nuclear RNA U6 was used as an\ninternal control and was amplified with the following\nprimers: forward, 5 ′-CTCGCTTCGGCAGCACA-3′;r e -\nverse, 5 ′-AACGCTTCACGAATTTGCGT-3′. The fol-\nlowing primers were also used for the indicated genes:\nFOXO1A, 5 ′-CCTCTGGATTGAGCATCCAC-3 ′ and\n5′-ATGTATGGAGGTGGGTCAGC-3′;P R L ,5 ′-CACT\nACATCCATAACCTCTC-3′ and 5 ′-ATGCTGACTATC\nAAGCTCAG-3′;I G F B P 1 ,5′-TATGATGGCTCGAAGG\nCTCTC-3′ and 5 ′-GTAGACGCACCAGCAGAGTC-3′;\nDCN, 5 ′-AGCTCTCCTACATCCGCATT-3′ and 5 ′-GC\nTAGCTGCATCAACTCTGC-3′; TIMP3, 5 ′-TGACAGG\nTCGCGTCTATGAT-3′ and 5 ′-CAACCCAGGTGATAC\nCGATAG-3′;K L F 1 2 ,5 ′-CCTTTCCATAGCCAGAGCA\nG-3′ and 5 ′-TTGCATCCCTCAAAATCACA-3 ′;1 8 S\nrRNA, 5 ′-CGGCTACCACATCCAAGGAA-3′ and 5 ′-C\nTGGAATTACCGCGGCT-3′. Samples were run in dupli-\ncate with RNA preparations from three independent ex-\nperiments. The fold change in expression of each gene\nwas calculated using the 2 -△△CT method, and 18S rRNA or\nU6 served as an internal control.\nWestern blotting\nBriefly, protein extracts were prepared from hESC as\npreviously described [23]. Equal amounts of total protein\n(30 μg) were separated on a 10% SDS-polyacrylamide gel\nand transferred onto polyvinylidene fluoride membranes\n(Millipore, Billerica, MA, USA). Immunoblotting was per-\nformed with primary antibodies against KLF12 (1:500;\nSanta Cruz Biotechnology, Santa Cruz, CA, USA) or β-\nactin (1:10,000; Abcam, Cambridge, MA, USA) followed\nby a goat anti-rabbit HRP-conjugated secondary antibody\n(1:10,000; Bioworld Technology, St. Louis Park, MN,\nUSA). Bands were detected using an enhanced chemilu-\nminescence kit (Amersham Biosciences Corp., Piscataway,\nNJ, USA).\nLuciferase reporter assay\nThe sequence (5 ′-CTGCGTATAAGGGACTGAATGTG\nAGGTAACTCTTATG-3′) in the 3 ′UTR of the human\nKLF12 gene containing the miR-181a seed sequence\nTGAATGT (pmirGLO-KLF12 3 ′UTR) or the sequence\n(CTGCGTATAAGGGAC GAGGTAACTCTTATG) that\nlacks TGAATGT (pmirGLO-KLF12 3 ′UTR mut) were\nsubcloned in the pmirGLO Dual-Luciferase miRNA\nTarget Expression Vector (pmirGLO vector, Promega,\nMadison, WI, USA). Preconfluent (70%) hESC in six-\nwell plates were infected with Ad-miR-181a and then\ntransfected with 300 ng of the luciferase reporter plas-\nmids using Lipofectamine 2000 for 48 h. The cell lysates\nwere assayed for luciferase activity using the Luciferase\nAssay System (Promega, Madison, WI, USA), and the\nactivity was measured using a luminescence counter\n(Centro XS3 LB 960, Berthold Technologies).\nImmunofluorescence staining for F-actin filaments\nhESC were grown in 8-well chambers (Millipore, Billerica,\nMA, USA) and fixed with 4% paraformaldehyde for\n30 min at room temperature, permeabilized with 0.5%\nTriton X-100 in PBS, and incubated with Alexa Fluor\n594-conjugated phalloidin for F-actin filaments staining\n(Sigma) at 4°C overnight. The cell nuclei were stained\nwith DAPI (5 μg/mL) on the following day. Finally, im-\nages were visualized using a fluorescence microscope\n(Leica, Wetzlar, Germany).\nProlactin examination by enzyme-linked fluorescent assay\n(ELFA)\nProlactin levels in the supernatant of hESC cultured with\nphenol red-free DMEM/F12 medium containing 2.5%\ncharcoal/dextran-treated FBS were measured using the\nMini-Vidas V.B. 02.96 system with Vidas prolactin kits\n(bioMérieux, France). The limit of detection of this kit\nwas 0.5 ng/mL.\nStatistical analysis\nAll experiments were performed at least three times.\nStatistical analysis was performed by ANOVA, followed\nby Student-Newman-Keuls tests for experiments involving\nmore than two groups. Student ’ s t-tests were performed\nfor comparisons between two groups. p-values <0.05 were\nconsidered to be statistically significant.\nResults\nEnhanced expression of miR-181a induces hESC\ndecidualization in vitro\nmiR-181a expression pattern was investigated in hESC\ntreated with 8-Br-cAMP and MPA for different periods\nof time (3, 6, 12, 24, and 48 h, respectively). qRT-PCR\nresults showed that miR-181a expression was increased\nafter 8-Br-cAMP and MPA treatment, and most effi-\nciently elevated at 6 h time point (Figure 1A). To investi-\ngate whether miR-181a regulates hESC decidualization,\nwe overexpressed miR-181a using adenoviral technology\n(Figure 1B). Then we tested decidualization-related gene\nexpression and observed that adenovirus-mediated over-\nexpression of miR-181a in hESC markedly increased\nFOXO1A mRNA expression and the expression of its\nZhang et al. Reproductive Biology and Endocrinology  (2015) 13:23 Page 3 of 9\n\ntargeted genes (PRL, IGFBP-1, DCN, and TIMP3)\n(Figure 1C – G). In addition, when 8-Br-cAMP and\nMPA were added to miR-181a-overexpressing cells,\nthe levels of FOXO1A, PRL, IGFBP-1, DCN, and TIMP3\nwere higher than those observed with Ad-miR-181a or\n8-Br-cAMP and MPA treatment alone (Figure 1C – G).\nMoreover, miR-181a overexpression significantly in-\ncreased decidual PRL secretion in a time-dependent\nmanner following the stimulation of decidualization\n(Figure 1H).\nBecause the decidualization of hESC is also charac-\nterized by the transformation of fibroblast-like hESC\ninto a round, epithelioid shape, we further examined\nwhether miR-181a affects the organization of the F-actin\ncytoskeleton. As shown in Figure 1I, decidualized hESC\ntreated with 8-Br-cAMP and MPA displayed more pol-\nygonal cell morphology with a random distribution of\nF-actin filaments compared with non-decidualized hESC.\nIn the absence of exogenous hormones, miR-181a overex-\npression caused the long, fibroblast-like shape of hESC to\nbecome noticeably rounder and the actin filaments to re-\narrange without direction.\nmiR-181a inhibition compromises hESC decidualization\nin vitro\nWe transfected hESC with a synthesized anti-sense\noligonucleotide of miR-181a (miR-181a inhibitor) to cor-\nroborate its regulatory effect on decidualization markers.\nFigure 1 miR-181a induces hESC decidualization in vitro . (A) Expression pattern of miR-181a in hESC treated with 0.5 mM 8-Br-cAMP and\n1 μM MPA (8Br + MPA) for different periods of time (3, 6, 12, 24, and 48 h, respectively) was evaluated by qRT-PCR. *p < 0.05, **p < 0.01. hESC were\ninfected with Ad-miR-181a or Ad-LacZ (MOI = 100). After 24 h, these cells were treated with 0.5 mM 8-Br-cAMP and 1 μM MPA as indicated for an\nadditional 72 h. miR-181a (B), FOXO1A (C), PRL (D), IGFBP1 (E), DCN (F), and TIMP3 (G) mRNA levels were measured by qRT-PCR. **p < 0.01, bars\nlabeled with different letters indicate statistically significant differences (p < 0.05).(H) hESC were infected with Ad-miR-181a or Ad-LacZ (MOI = 100) for\n24 h followed by treatment with 0.5 mM 8-Br-cAMP and 1 μM MPA for the indicated times. Prolactin released into the medium was detected by ELFA.\n*p < 0.05, **p < 0.01, compared with Ad-LacZ treated with 8-Br-cAMP and MPA. (I) Immunofluorescence using Alexa Fluor 594-conjugated phalloidin\nto label actin filaments was performed to analyze the morphological transformation of hESC.\nZhang et al. Reproductive Biology and Endocrinology  (2015) 13:23 Page 4 of 9\n\nmiR-181a inhibitor specifically suppressed endogenous\nmiR-181a expression without affecting miR-181b, miR-\n181c, or miR-181d levels in hESC (Figure 2A). Inhibition\nof miR-181a in hESC led to a significant decrease in\nFOXO1A, PRL, IGFBP-1, DCN, and TIMP3 gene expres-\nsion induced by 8-Br-cAMP and MPA (Figure 2B – F). Fur-\nthermore, decidualized hESC reverted from a round,\nepithelioid-like morphology to a fibroblast-like phenotype\nafter miR-181a inhibitor transfection (Figure 2G).\nmiR-181a inhibits KLF12 expression\nWe next sought to identify the potential target driving\nmiR-181a-mediated hESC decidualization in vitro , and\nfocused on KLF12 gene, which is down-regulated in\ndecidualized hESC [22]. Based on the mouse and human\nKLF12 mRNA sequences deposited in GenBank (NCBI\nReference Sequences: NM_010636.3 and NM_007249.4),\nwe found a miR-181a seed target region within the\nKLF12 mRNA 3 ′UTR (Figure 3A). To verify that KLF12\nis a potential target of miR-181a, a luciferase-based re-\nporter assay was performed using the KLF12 3 ′UTR. In-\ncreased expression of miR-181a in hESC significantly\ndecreased luciferase reporter activity by approximately\n50% (Figure 3B). A mutated KLF12 3 ′UTR in which\nseven nucleotides of the miR-181a binding site were\nabolished no longer responded to miR-181a modulation\n(Figure 3B).\nConsistent with KLF12 3 ′UTR reporter results, miR-\n181a overexpression in hESC significantly down-regulated\nendogenous levels of KLF12 at both the mRNA and pro-\ntein levels. Reduced miR-181a expression resulted in the\nup-regulation of KLF12 mRNA and protein expression in\nhESC (Figure 3C and D).\nKLF12 overexpression attenuates miR-181a-mediated\ndecidualization events\nFinally, we assessed whether KLF12 is involved in the\nprocess of miR-181a-induced hESC decidualization.\nFigure 2 miR-181a inhibition attenuates hESC decidualization in vitro. hESC were transfected with a miR-181a inhibitor or a miRNA inhibitor\nnegative control (miRNA inhibitor control, 100 nM) for 24 h, and then cells were treated with or without 0.5 mM 8-Br-cAMP and 1 μM MPA\n(8Br + MPA) for an additional 72 h. miR-181 family (A), FOXO1A (B), PRL (C), IGFBP1 (D), DCN (E), and TIMP3 (F) mRNA expression levels were\nexamined by qRT-PCR. *p < 0.05, bars labeled with different letters indicate statistically significant differences (p < 0.05). (G) The decidual\ntransformation change of hESC based on the distribution of the actin filaments.\nZhang et al. Reproductive Biology and Endocrinology  (2015) 13:23 Page 5 of 9\n\nAdenovirus-mediated KLF12 overexpression (Figure 4A)\nin hESC suppressed the mRNA expression of FOXO1A,\nPRL, IGFBP-1, DCN, and TIMP3, compared to Ad-LacZ\ngroup (Figure 4B – F). KLF12 overexpression also attenu-\nated miR-181a-enhanced mRNA expression of these genes\n(Figure 4B – F). Moreover, KLF12 overexpression blocked\nmiR-181a-induced epithelioid-like morphological changes\nof hESC (Figure 4G).\nDiscussion\nThe transformation of endometrium into decidua is\nessential for normal implantation of the blastocyst, a\nprocess in which many key proteins and growth factors\nplay fundamental roles [2,3]. The participation of estrogen\nand progesterone is vital for stromal cell decidualization,\nas progesterone receptor or estrogen receptor knockout\nmice both fail to display endometrial decidualization and\nas 8-Br-cAMP and MPA treatment can induce hESC\ndecidualization in vitro [22,25,26]. miRNAs are also in-\nvolved in this process, although their exact role in normal\nembryonic formation, endometrial preparation for preg-\nnancy, and decidualization remains unclear [11,13,15].\nHere, we found that miR-181a level is increased in the\nprocess of 8-Br-cAMP and MPA-induced hESC deciduali-\nzation in vitro , suggesting that miR-181a may play func-\ntions in this process.\nmiR-181a has been demonstrated to be a key modulator\nof cellular differentiation, including hematopoietic lineages\nand myoblasts, as well as T-cell sensitivity and selection\n[27-29]. Recently, we identified that miR-181a suppresses\nmouse granulosa cell proliferation by targeting activin re-\nceptor IIA (acvr2a) and thus regulates activin-induced\ngene expression [24]. In this study, our data confirmed\nthat miR-181a promotes decidu alization-related gene\nFigure 3 miR-181a inhibits KLF12 expression. (A) Putative miR-181a-targeting sites in the human and mouse KLF12 mRNA 3 ′UTRs. (B) Analysis\nof miR-181a modulation on wild-type or mutant KLF12 3 ′UTR luciferase reporter plasmids. **p < 0.01 (n = 3). hESC were infected with Ad-miR-181a\nor transfected with miR-181a inhibitor as indicated for 48 h. qRT-PCR and western blotting were performed to examine endogenous KLF12 mRNA\n(C) and protein (D) expression. *p < 0.05, compared with control group, **p < 0.01, compared with the Ad-LacZ group.\nZhang et al. Reproductive Biology and Endocrinology  (2015) 13:23 Page 6 of 9\n\nexpression and causes a noticeable change in stromal\ncell shape. Furthermore, miR-181a inhibition causes an\nimpaired induction of the decidual reaction by 8-Br-\ncAMP and MPA. These results demonstrate that miR-\n181a plays a positive role in hESC decidualization. Other\nrecent studies support the crucial roles that miRNAs play\nin decidualization. For example, Dicer mRNA and protein\nlevels were significantly up-regulated after decidualization\ntreatment using cAMP and MPA [11]. miR-222 regulates\nhESC differentiation by targeting CDKN1C/p57kip2 ex-\npression [30]. miR-135b also targets HOXA10, which is\nessential for female fertility and decidualization [31,32].\nMoreover, miR-141, miR-143, and miR-193 are differently\nexpressed in the mouse uteri before and after embryo im-\nplantation [33-35]. miR-181a is also reported to be highly\nexpressed in the porcine endometrium on day 15 gesta-\ntion, compared to that on day 26 and 50 gestation [15].\nHowever, the function of miR-181a in endometrial decid-\nualization is unclear. Our study confirms that miR-181a\ninduces hESC decidualization.\nThe effects of progesterone are mediated through\ninteractions with the progesterone receptor (PGR). PGR\nphysically associates with other nuclear transcription\nfactors, such as FOXO1A and the estrogen receptor, to\nregulate decidualization-specific gene expression [36,37].\nThe transcription factor FOXO1A is critical for decidua-\nlization and promotes the expression of decidualization-\nassociated targeted genes, such as PRL, IGFBP-1, DCN,\nand TIMP3 during the decidualization process [38,39].\nThe transcriptional ability of FOXO1A is regulated by\nmany factors in the process of endometrial decidualiza-\ntion, such as PI3K/Akt, PGR, and HoxA10 [40-42]. To\ndate, the function of miRNAs involved in regulating\nFOXO1A expression and activation is largely uncovered.\nIn this study, we revealed that overexpression of miR-181a\nincreases FOXO1A mRNA expression and miR-181a in-\nhibitor suppresses 8-Br-cAMP and MPA-induced FOXO1A\nexpression, indicating that miR-181a mediates the promot-\ning effect of decidual stimuli on FOXO1A expression.\nTo identify physiological targets of miR-181a involved\nin the process of miR-181a-induced decidual gene expres-\nsion, we focused on KLF12, a novel transcription factor\nidentified by our laboratory that negatively regulates hESC\ndecidualization [22]. A luciferase assay demonstrated that\nFigure 4 KLF12 overexpression represses the miR-181a-induced morphological and biochemical transformation of hESC. hESC were\ninfected with Ad-miR-181a (100 MOI) and/or Ad-KLF12 (20 MOI) as indicated for 72 h. KLF12 (A), FOXO1A (B), PRL (C), IGFBP1 (D), DCN (E), and\nTIMP3 (F) mRNA levels were measured by qRT-PCR. **p < 0.01, bars labeled with different letters indicate statistically significant differences (p < 0.05).\n(G) The morphological changes of hESC were detected by immunofluorescence staining.\nZhang et al. Reproductive Biology and Endocrinology  (2015) 13:23 Page 7 of 9\n\nmiR-181a interacts with the 3 ′UTR of KLF12 and down-\nregulates KLF12 at the transcriptional and translational\nlevels. Re-expression of KLF12 abolished miR-181a-\ninduced decidualization, suggesting that KLF12 is a critical\nmediator of miR-181a-induced decidualization. Members\nof the KLF family of zinc-finger transcription factors are\ncritical for the development of uterine receptivity and the\ndifferentiation of stromal cells [19,20]. KLF12 protein is\nsignificantly decreased after the stimulation of deciduali-\nzation, and KLF12 overexpression in hESC significantly\nrepresses the expression of decidualization marker genes\nand cell morphology changes [22]. Interestingly, based\non the human FOXO1A promoter sequence (accession\nno: 11424), the dPRL promoter sequence (accession no:\n37139), and the IGFBP-1 prom oter sequence (accession\nno: 37680) deposited in the Transcriptional Regulatory\nElement Database [43], we found conserved CAGTGGG\nelements within the promoter core regions of these\ngenes, suggesting the possibility of a direct role for\nKLF12 in regulating their expression. In the future, we\nwill further study the molecular mechanisms of miR-\n181a and KLF12 in decidualization.\nAberrant miRNA expression is associated with a wide\nvariety of human diseases. Endometrial miR-181a and\nmiR-98 are aberrantly expressed in endometrial tumors\n[44], and miR-181a plays a critical role in epithelial ovar-\nian cancer (EOC) progression through the regulation of\nthe epithelial-mesenchymal transition by modulating the\nTGF-β signaling pathway [45]. We recently found that\nKLF12 was markedly up-regulated in endometrium from\nendometriosis and RIF patients (unpublished data), and\nwe will further investigate the expression patterns of\nmiR-181a in these patients to better understand the role\nmiR-181a plays in the pathogenesis of these diseases.\nConclusions\nTogether, this study highlights a novel role of miR-181a\nand KLF12 in the decidualization process of human\nendometrial stromal cell. Our findings provide novel po-\ntential biomarkers and therapeutic targets for diseases\nassociated with defective decidualization.\nAbbreviations\nacvr2a: Activin receptor IIA; CREB1/3: cAMP response element binding\nprotein 1/3; DCN: Decorin; Esr1: Estrogen receptor 1; ETS1: v-ets\nerythroblastosis virus E26 oncogene homolog 1; FOXO1A: Forkhead\nheadbox O1A; hESC: Human endometrial stromal cell; HOXA10: Homeobox\nA10; IGFBP-1: Insulin-like growth factor binding protein-1; IL-1:\nInterleukin-1; KLF12: Krüppel-like factor 12; miR-181a: microRNA-181a;\nMPA: Medroxyprogesterone acetate; PR: Progesterone receptor; PRL: Prolactin;\nRIF: Repeated implantation failure; TNF-α: Tumor necrosis factor-α;3 ′UTR: 3′\nuntranslated region; 8-Br-cAMP: 8-bromoadenosine-cAMP.\nCompeting interests\nThe authors declare that they have no competing interests.\nAuthors’ contributions\nQZ, HZ, and YJ carried out the molecular genetics studies. XZ contributed to\nthe isolation of hESC. QZ and GY drafted the manuscript. BX, ZD, LD, and HS\nparticipated in the design of the study and performed the statistical analysis.\nGY and YH conceived of the study, and participated in its design. All authors\nread and approved the final manuscript.\nAcknowledgments\nThis work was supported by the National Natural Science Foundation of\nChina (81370683, 81170570, 81370724, and 81070492), a special grant for\nprincipal investigators from the Health Department of Jiangsu Province\n(LJ201102 and RC2011005) and the program for the six top talents of\nJiangsu Province, PR China (No. 2012-WSN-005).\nAuthor details\n1Reproductive Medicine Center, Department of Obstetrics and Gynecology,\nNanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing\n210008, Jiangsu, China. 2Reproductive Medicine Center, Drum Tower Clinic\nMedical College of Nanjing Medical University, Nanjing 210029, Jiangsu,\nChina.\nReceived: 11 November 2014 Accepted: 14 March 2015\nReferences\n1. 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Parikh A, Lee C, Peronne J, Marchini S, Baccarini A, Kolev V, et al. microRNA-181a\nhas a critical role in ovarian cancer progression through the regulation of the\nepithelial-mesenchymal transition. Nat Commun. 2014;5:2977.\nSubmit your next manuscript to BioMed Central\nand take full advantage of: \n• Convenient online submission\n• Thorough peer review\n• No space constraints or color figure charges\n• Immediate publication on acceptance\n• Inclusion in PubMed, CAS, Scopus and Google Scholar\n• Research which is freely available for redistribution\nSubmit your manuscript at \nwww.biomedcentral.com/submit\nZhang et al. Reproductive Biology and Endocrinology  (2015) 13:23 Page 9 of 9","source_license":"CC0","license_restricted":false}