MicroRNA-181a is involved in the regulation of human endometrial stromal cell decidualization by inhibiting Krüppel-like factor 12

Background: The transformation of endometrium into decidua is essential for normal implantation of the blastocyst. However, the post-transcriptional regulation and the miRNAs involved in decidualization remain poorly understood. Here, we examined microRNA-181a (miR-181a) expression in decidualized human endometrial stromal cell (hESC). In addition, we investigated the functional effect of miR-181a on hESC decidualization in vitro.

Quantitative real-time PCR (qRT-PCR) was used to de tect the profile of miR-181a in decidualized hESC. qRT-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 marker genes’ expression after enhancing or inhibition of miR-181a expression in hESC. Luciferase reporter assay, western blotting, qRT-PCR, and immunofluorescence assay were carried out to identify the relationship between miR-181a and Krüppel-like factor 12 (KLF12).

miR-181a expression levels increased dramatically in hESC treated with 8-Br-cAMP and MPA. Increased miR-181a expression promoted hESC decidualization-related gene e xpression and morphological transformation; conversely, inhibition of miR-181a expression compromised hESC decidualization in vitro . Further analysis confirmed that miR-181a interacted with the 3 ′ untranslated region of the transcription factor KLF12 and down-regulated KLF12 at the transcriptional and translational l evels. KLF12 overexpression abolishe d miR-181a-induced decidualization.

Our findings suggest that miR-181a plays a functionally important role in human endometrial s 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.

MicroRNA-181a, KLF12, Human endometr ial stromal cell, Decidualization

Decidualization of the endometrial stroma is a precondi- tion for the successful establishment of pregnancy. In humans, this process is initiated in the mid-secretory phase of the menstrual cycle and is triggered by ovarian sex steroid hormones independent of pregnancy [1,2]. The decidual reaction consists of a dramatic morpho- logical and biochemical transformation of the endomet- rial stroma in which the stromal fibroblasts differentiate to become rounded, relatively large epithelioid-like or polygonal, secretory decidual cells [3]. Human decidual cells produce specific molecules, such as regulatory fac- tors (prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1)), inflammatory mediators (IL-1, IL-6, IL-8, and TNF- α), and specific extracellular matrix proteins (laminin, type IV collagen, and fibronec- tin) [4-7]. PRL and IGFBP-1 levels are generally used as biochemical decidualization markers of progestin-induced human endometrial stromal cell (hESC) differentiation. A number of transcription factors and autocrine/paracrine factors have been identified that cooperatively control the decidualization process. However, little information is available regarding the post-transcriptional regulation of this process. * Correspondence: [email protected]; [email protected] † Equal contributors 1Reproductive Medicine Center, Department of Obstetrics and Gynecology, Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing 210008, Jiangsu, China Full list of author information is available at the end of the article © 2015 Zhang et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhang et al. Reproductive Biology and Endocrinology (2015) 13:23 DOI 10.1186/s12958-015-0019-y MicroRNAs (miRNAs) have emerged as key post- transcriptional regulators. An estimated 30 – 50% of protein-coding genes serve as potential miRNA targets. miRNAs regulate and influence a variety of cellular ac- tivities, including cell growth, differentiation, apoptosis, and metabolism [8]. miRNAs are small (approximately 20– 22 nt), noncoding RNAs that generally base-pair within the 3 ′ untranslated region (3 ′UTR) of target mRNAs, causing translational inhibition and/or mRNA degradation [9]. Recently, the conditional inactivation of Dicer has provided evidence for the pivotal functions of miRNAs in ovarian as well as oviductal and uterine stro- mal cell development [10]. Dicer expression increases and is a requirement during human endometrial stromal decidualization in vitro [11]. The aberrant expression of some miRNAs has been correlated with various endomet- rial diseases, such as endometriosis, repeated implantation failure (RIF), and endometrial cancer [12-14]. MicroRNA-181a (miR-181a), which belongs to the miR-181 family, is a key modulator of cellular differenti- ation. Based on microRNA microarray analysis, L. Su et al. found high miR-181a/c expression on day 15 of gestation, followed by decreased expression on gesta- tional days 26 and 50 in the porcine endometrium dur- ing pregnancy [15]. Various potential miR-181 family targets, such as ETS1, CREB1/3, Esr1, and PGR, are in- volved during differentiation and decidualization events [16-18]. The Krüppel-like factor (KLF) family members are revealed to play critical roles in regulating the process of embryo implantation, such as KLF9 and KLF13 [19,20]. KLF12, another member of KLF family, binds to the CAGTGGG sequence within target gene promoter regions and represses target gene expression through an N-terminal PVDLS sequence (Pro-Xaa-Asp- Leu-Ser) that promotes a physical interaction with the co-repressor CtBPs [21]. We previously demonstrated that KLF12, suppressed by 8-Br-cAMP and MPA, nega- tively regulates hESC decidualization by inhibiting PRL and IGFBP-1 expression [22]. In this study, we demon- strated that miR-181a was involved in the regulation of hESC decidualization by suppressing KLF12, whereas KLF12 overexpression inhibited miR-181a-mediated in- creases in decidualization-related gene expression and the morphological transformation of hESC, indicating that miR-181a may play an important role in human endometrial decidualization.

Isolation and in vitro decidualization of hESC This study was approved by the Institutional Review Board of the Drum Tower Hospital of Nanjing University on December 5, 2013 (2013-081-01). This study was con- ducted in the Drum Tower Hospital from February 2014 to September 2014. Patient consent was received before biopsy. hESC were isolated from the mid-secretory phase of endometrial tissue of women with a normal menstrual cycle by endometrial biopsy. hESC were isolated and cul- tured as previously described [23]. To induce decidualization, hESC were cultured in phenol red-free DMEM/F12 medium (HyClone, Thermo Scientific, South Logan, UT, USA) containing 2.5% charcoal/dextran-treated fetal bovine serum (FBS, HyClone, Thermo Scientific, South Logan, UT, USA), 100 IU/ml penicillin, and 100 μg/ml of streptomycin supple- mented with 0.5 mM 8-Br-cAMP and 1 μMM P A (Sigma, St. Louis, MO, USA) for 3 days, 6 days, or 9 days. Differentiation was assessed by examination of cell morphology under phase contrast microscopy at various times during the treatment and also by meas- uring the expression of decidu alization-specific marker gene, namely PRL. Construction of adenovirus To overexpress KLF12 and miR-181a in hESC, adenovirus vectors harboring the full-length KLF12 (Ad-KLF12) and precursor miR-181a (Ad-miR-181a) were generated using the AdMax (Microbix Biosystems, Inc., Toronto, Canada) and pSilencer ™ adeno 1.0-CMV (Ambion, Austin, TX, USA) systems as previously described [22,24]. The primers for full-length KLF 12 amplification were: 5 ′-T CTCGAATTCAATGAATATCCATATGAAGAG-3′ and 5′-TATAGGATCCTCACACCAACATATGCCTCC-3′; for precursor miR-181a amplification were: 5 ′-CGCGC TCGAGATACAATGTGATGTGGAGGTT-3′ and 5 ′-G CGCGATATCGGCCACAGTTGCATTCATTGT-3′.A n adenovirus bearing LacZ (Ad-LacZ) was obtained from Clontech (Palo Alto, CA, USA) and used as the control of adenovirus infection experiments. The viruses were packaged and amplified in HEK293A cells and purified using CsCl banding followed by dialysis against 10 mM Tris-buffered saline with 10% glycerol. The viral titer was determined using HEK293A cells and the Adeno-X Rapid Titer kit (Clontech). hESC were infected with Ad-miR-181a at the 100 MOI and/or with Ad-KLF12 at the 20 MOI. miRNA inhibitor transfection miR-181a inhibitor (2 ′-O-methyl modified oligonu- cleotides: 5 ′-mAmCmUmCmAmCmCmGmAmCmAmG mCmGmUmUmGmAmAmUmGmUmU-3 ′)o rm i R N A inhibitor negative control (miRNA inhibitor control) were synthesized by Ribobio (Guangzhou, China). miRNA inhibitor control shares no homologous region with the human genome sequences. For loss of function experiments, hESC were transfected with 100 nM of miR-181a inhibitor or of miRNA inhibitor control using Lipofectamine 2000 (Life Technologies, New York, USA). Zhang et al. Reproductive Biology and Endocrinology (2015) 13:23 Page 2 of 9 RNA isolation and quantitative real-time PCR (qRT-PCR) Total RNA was extracted from hESC using Trizol reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription was performed using random primers or specific miRNA stem-loop primers, and qRT-PCR was performed on a MyiQ Single-Color Real-Time PCR Detection System (BIO-RAD, Hercules, CA, USA). To detect miR-181a ex- pression, we used the following primers: forward, 5 ′-AC ACTCCAGCTGGGAACATTCAACGCTGTCG-3 ′;r e - verse, 5 ′-GGTGTCGTGGAGTCGGCAATTCAGTTG AG-3′. The small nuclear RNA U6 was used as an internal control and was amplified with the following primers: forward, 5 ′-CTCGCTTCGGCAGCACA-3′;r e - verse, 5 ′-AACGCTTCACGAATTTGCGT-3′. The fol- lowing primers were also used for the indicated genes: FOXO1A, 5 ′-CCTCTGGATTGAGCATCCAC-3 ′ and 5′-ATGTATGGAGGTGGGTCAGC-3′;P R L ,5 ′-CACT ACATCCATAACCTCTC-3′ and 5 ′-ATGCTGACTATC AAGCTCAG-3′;I G F B P 1 ,5′-TATGATGGCTCGAAGG CTCTC-3′ and 5 ′-GTAGACGCACCAGCAGAGTC-3′; DCN, 5 ′-AGCTCTCCTACATCCGCATT-3′ and 5 ′-GC TAGCTGCATCAACTCTGC-3′; TIMP3, 5 ′-TGACAGG TCGCGTCTATGAT-3′ and 5 ′-CAACCCAGGTGATAC CGATAG-3′;K L F 1 2 ,5 ′-CCTTTCCATAGCCAGAGCA G-3′ and 5 ′-TTGCATCCCTCAAAATCACA-3 ′;1 8 S rRNA, 5 ′-CGGCTACCACATCCAAGGAA-3′ and 5 ′-C TGGAATTACCGCGGCT-3′. Samples were run in dupli- cate with RNA preparations from three independent ex- periments. The fold change in expression of each gene was calculated using the 2 -△△CT method, and 18S rRNA or U6 served as an internal control. Western blotting Briefly, protein extracts were prepared from hESC as previously described [23]. Equal amounts of total protein (30 μg) were separated on a 10% SDS-polyacrylamide gel and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Immunoblotting was per- formed with primary antibodies against KLF12 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or β- actin (1:10,000; Abcam, Cambridge, MA, USA) followed by a goat anti-rabbit HRP-conjugated secondary antibody (1:10,000; Bioworld Technology, St. Louis Park, MN, USA). Bands were detected using an enhanced chemilu- minescence kit (Amersham Biosciences Corp., Piscataway, NJ, USA). Luciferase reporter assay The sequence (5 ′-CTGCGTATAAGGGACTGAATGTG AGGTAACTCTTATG-3′) in the 3 ′UTR of the human KLF12 gene containing the miR-181a seed sequence TGAATGT (pmirGLO-KLF12 3 ′UTR) or the sequence (CTGCGTATAAGGGAC GAGGTAACTCTTATG) that lacks TGAATGT (pmirGLO-KLF12 3 ′UTR mut) were subcloned in the pmirGLO Dual-Luciferase miRNA Target Expression Vector (pmirGLO vector, Promega, Madison, WI, USA). Preconfluent (70%) hESC in six- well plates were infected with Ad-miR-181a and then transfected with 300 ng of the luciferase reporter plas- mids using Lipofectamine 2000 for 48 h. The cell lysates were assayed for luciferase activity using the Luciferase Assay System (Promega, Madison, WI, USA), and the activity was measured using a luminescence counter (Centro XS3 LB 960, Berthold Technologies). Immunofluorescence staining for F-actin filaments hESC were grown in 8-well chambers (Millipore, Billerica, MA, USA) and fixed with 4% paraformaldehyde for 30 min at room temperature, permeabilized with 0.5% Triton X-100 in PBS, and incubated with Alexa Fluor 594-conjugated phalloidin for F-actin filaments staining (Sigma) at 4°C overnight. The cell nuclei were stained with DAPI (5 μg/mL) on the following day. Finally, im- ages were visualized using a fluorescence microscope (Leica, Wetzlar, Germany). Prolactin examination by enzyme-linked fluorescent assay (ELFA) Prolactin levels in the supernatant of hESC cultured with phenol red-free DMEM/F12 medium containing 2.5% charcoal/dextran-treated FBS were measured using the Mini-Vidas V.B. 02.96 system with Vidas prolactin kits (bioMérieux, France). The limit of detection of this kit was 0.5 ng/mL. Statistical analysis All experiments were performed at least three times. Statistical analysis was performed by ANOVA, followed by Student-Newman-Keuls tests for experiments involving more than two groups. Student ’ s t-tests were performed for comparisons between two groups. p-values <0.05 were considered to be statistically significant.

Enhanced expression of miR-181a induces hESC decidualization in vitro miR-181a expression pattern was investigated in hESC treated with 8-Br-cAMP and MPA for different periods of time (3, 6, 12, 24, and 48 h, respectively). qRT-PCR

showed that miR-181a expression was increased after 8-Br-cAMP and MPA treatment, and most effi- ciently elevated at 6 h time point (Figure 1A). To investi- gate whether miR-181a regulates hESC decidualization, we overexpressed miR-181a using adenoviral technology (Figure 1B). Then we tested decidualization-related gene expression and observed that adenovirus-mediated over- expression of miR-181a in hESC markedly increased FOXO1A mRNA expression and the expression of its Zhang et al. Reproductive Biology and Endocrinology (2015) 13:23 Page 3 of 9 targeted genes (PRL, IGFBP-1, DCN, and TIMP3) (Figure 1C – G). In addition, when 8-Br-cAMP and MPA were added to miR-181a-overexpressing cells, the levels of FOXO1A, PRL, IGFBP-1, DCN, and TIMP3 were higher than those observed with Ad-miR-181a or 8-Br-cAMP and MPA treatment alone (Figure 1C – G). Moreover, miR-181a overexpression significantly in- creased decidual PRL secretion in a time-dependent manner following the stimulation of decidualization (Figure 1H). Because the decidualization of hESC is also charac- terized by the transformation of fibroblast-like hESC into a round, epithelioid shape, we further examined whether miR-181a affects the organization of the F-actin cytoskeleton. As shown in Figure 1I, decidualized hESC treated with 8-Br-cAMP and MPA displayed more pol- ygonal cell morphology with a random distribution of F-actin filaments compared with non-decidualized hESC. In the absence of exogenous hormones, miR-181a overex- pression caused the long, fibroblast-like shape of hESC to become noticeably rounder and the actin filaments to re- arrange without direction. miR-181a inhibition compromises hESC decidualization in vitro We transfected hESC with a synthesized anti-sense oligonucleotide of miR-181a (miR-181a inhibitor) to cor- roborate its regulatory effect on decidualization markers. Figure 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 1 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 infected 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 additional 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 labeled with different letters indicate statistically significant differences (p < 0.05).(H) hESC were infected with Ad-miR-181a or Ad-LacZ (MOI = 100) for 24 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. *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 to label actin filaments was performed to analyze the morphological transformation of hESC. Zhang et al. Reproductive Biology and Endocrinology (2015) 13:23 Page 4 of 9 miR-181a inhibitor specifically suppressed endogenous miR-181a expression without affecting miR-181b, miR- 181c, or miR-181d levels in hESC (Figure 2A). Inhibition of miR-181a in hESC led to a significant decrease in FOXO1A, PRL, IGFBP-1, DCN, and TIMP3 gene expres- sion induced by 8-Br-cAMP and MPA (Figure 2B – F). Fur- thermore, decidualized hESC reverted from a round, epithelioid-like morphology to a fibroblast-like phenotype after miR-181a inhibitor transfection (Figure 2G). miR-181a inhibits KLF12 expression We next sought to identify the potential target driving miR-181a-mediated hESC decidualization in vitro , and focused on KLF12 gene, which is down-regulated in decidualized hESC [22]. Based on the mouse and human KLF12 mRNA sequences deposited in GenBank (NCBI

Sequences: NM_010636.3 and NM_007249.4), we found a miR-181a seed target region within the KLF12 mRNA 3 ′UTR (Figure 3A). To verify that KLF12 is a potential target of miR-181a, a luciferase-based re- porter assay was performed using the KLF12 3 ′UTR. In- creased expression of miR-181a in hESC significantly decreased luciferase reporter activity by approximately 50% (Figure 3B). A mutated KLF12 3 ′UTR in which seven nucleotides of the miR-181a binding site were abolished no longer responded to miR-181a modulation (Figure 3B). Consistent with KLF12 3 ′UTR reporter results, miR- 181a overexpression in hESC significantly down-regulated endogenous levels of KLF12 at both the mRNA and pro- tein levels. Reduced miR-181a expression resulted in the up-regulation of KLF12 mRNA and protein expression in hESC (Figure 3C and D). KLF12 overexpression attenuates miR-181a-mediated decidualization events Finally, we assessed whether KLF12 is involved in the process of miR-181a-induced hESC decidualization. Figure 2 miR-181a inhibition attenuates hESC decidualization in vitro. hESC were transfected with a miR-181a inhibitor or a miRNA inhibitor negative 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 (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 examined by qRT-PCR. *p < 0.05, bars labeled with different letters indicate statistically significant differences (p < 0.05). (G) The decidual transformation change of hESC based on the distribution of the actin filaments. Zhang et al. Reproductive Biology and Endocrinology (2015) 13:23 Page 5 of 9 Adenovirus-mediated KLF12 overexpression (Figure 4A) in hESC suppressed the mRNA expression of FOXO1A, PRL, IGFBP-1, DCN, and TIMP3, compared to Ad-LacZ group (Figure 4B – F). KLF12 overexpression also attenu- ated miR-181a-enhanced mRNA expression of these genes (Figure 4B – F). Moreover, KLF12 overexpression blocked miR-181a-induced epithelioid-like morphological changes of hESC (Figure 4G).

The transformation of endometrium into decidua is essential for normal implantation of the blastocyst, a process in which many key proteins and growth factors play fundamental roles [2,3]. The participation of estrogen and progesterone is vital for stromal cell decidualization, as progesterone receptor or estrogen receptor knockout mice both fail to display endometrial decidualization and as 8-Br-cAMP and MPA treatment can induce hESC decidualization in vitro [22,25,26]. miRNAs are also in- volved in this process, although their exact role in normal embryonic formation, endometrial preparation for preg- nancy, and decidualization remains unclear [11,13,15]. Here, we found that miR-181a level is increased in the process of 8-Br-cAMP and MPA-induced hESC deciduali- zation in vitro , suggesting that miR-181a may play func- tions in this process. miR-181a has been demonstrated to be a key modulator of cellular differentiation, including hematopoietic lineages and myoblasts, as well as T-cell sensitivity and selection [27-29]. Recently, we identified that miR-181a suppresses mouse granulosa cell proliferation by targeting activin re- ceptor IIA (acvr2a) and thus regulates activin-induced gene expression [24]. In this study, our data confirmed that miR-181a promotes decidu alization-related gene Figure 3 miR-181a inhibits KLF12 expression. (A) Putative miR-181a-targeting sites in the human and mouse KLF12 mRNA 3 ′UTRs. (B) Analysis of 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 or transfected with miR-181a inhibitor as indicated for 48 h. qRT-PCR and western blotting were performed to examine endogenous KLF12 mRNA (C) and protein (D) expression. *p < 0.05, compared with control group, **p < 0.01, compared with the Ad-LacZ group. Zhang et al. Reproductive Biology and Endocrinology (2015) 13:23 Page 6 of 9 expression and causes a noticeable change in stromal cell shape. Furthermore, miR-181a inhibition causes an impaired induction of the decidual reaction by 8-Br- cAMP and MPA. These results demonstrate that miR- 181a plays a positive role in hESC decidualization. Other recent studies support the crucial roles that miRNAs play in decidualization. For example, Dicer mRNA and protein levels were significantly up-regulated after decidualization treatment using cAMP and MPA [11]. miR-222 regulates hESC differentiation by targeting CDKN1C/p57kip2 ex- pression [30]. miR-135b also targets HOXA10, which is essential for female fertility and decidualization [31,32]. Moreover, miR-141, miR-143, and miR-193 are differently expressed in the mouse uteri before and after embryo im- plantation [33-35]. miR-181a is also reported to be highly expressed in the porcine endometrium on day 15 gesta- tion, compared to that on day 26 and 50 gestation [15]. However, the function of miR-181a in endometrial decid- ualization is unclear. Our study confirms that miR-181a induces hESC decidualization. The effects of progesterone are mediated through interactions with the progesterone receptor (PGR). PGR physically associates with other nuclear transcription factors, such as FOXO1A and the estrogen receptor, to regulate decidualization-specific gene expression [36,37]. The transcription factor FOXO1A is critical for decidua- lization and promotes the expression of decidualization- associated targeted genes, such as PRL, IGFBP-1, DCN, and TIMP3 during the decidualization process [38,39]. The transcriptional ability of FOXO1A is regulated by many factors in the process of endometrial decidualiza- tion, such as PI3K/Akt, PGR, and HoxA10 [40-42]. To date, the function of miRNAs involved in regulating FOXO1A expression and activation is largely uncovered. In this study, we revealed that overexpression of miR-181a increases FOXO1A mRNA expression and miR-181a in- hibitor suppresses 8-Br-cAMP and MPA-induced FOXO1A expression, indicating that miR-181a mediates the promot- ing effect of decidual stimuli on FOXO1A expression. To identify physiological targets of miR-181a involved in the process of miR-181a-induced decidual gene expres- sion, we focused on KLF12, a novel transcription factor identified by our laboratory that negatively regulates hESC decidualization [22]. A luciferase assay demonstrated that Figure 4 KLF12 overexpression represses the miR-181a-induced morphological and biochemical transformation of hESC. hESC were infected 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 TIMP3 (F) mRNA levels were measured by qRT-PCR. **p < 0.01, bars labeled with different letters indicate statistically significant differences (p < 0.05). (G) The morphological changes of hESC were detected by immunofluorescence staining. Zhang et al. Reproductive Biology and Endocrinology (2015) 13:23 Page 7 of 9 miR-181a interacts with the 3 ′UTR of KLF12 and down- regulates KLF12 at the transcriptional and translational levels. Re-expression of KLF12 abolished miR-181a- induced decidualization, suggesting that KLF12 is a critical mediator of miR-181a-induced decidualization. Members of the KLF family of zinc-finger transcription factors are critical for the development of uterine receptivity and the differentiation of stromal cells [19,20]. KLF12 protein is significantly decreased after the stimulation of deciduali- zation, and KLF12 overexpression in hESC significantly represses the expression of decidualization marker genes and cell morphology changes [22]. Interestingly, based on the human FOXO1A promoter sequence (accession no: 11424), the dPRL promoter sequence (accession no: 37139), and the IGFBP-1 prom oter sequence (accession no: 37680) deposited in the Transcriptional Regulatory Element Database [43], we found conserved CAGTGGG elements within the promoter core regions of these genes, suggesting the possibility of a direct role for KLF12 in regulating their expression. In the future, we will further study the molecular mechanisms of miR- 181a and KLF12 in decidualization. Aberrant miRNA expression is associated with a wide variety of human diseases. Endometrial miR-181a and miR-98 are aberrantly expressed in endometrial tumors [44], and miR-181a plays a critical role in epithelial ovar- ian cancer (EOC) progression through the regulation of the epithelial-mesenchymal transition by modulating the TGF-β signaling pathway [45]. We recently found that KLF12 was markedly up-regulated in endometrium from endometriosis and RIF patients (unpublished data), and we will further investigate the expression patterns of miR-181a in these patients to better understand the role miR-181a plays in the pathogenesis of these diseases.

Together, this study highlights a novel role of miR-181a and KLF12 in the decidualization process of human endometrial stromal cell. Our findings provide novel po- tential biomarkers and therapeutic targets for diseases associated with defective decidualization. Abbreviations acvr2a: Activin receptor IIA; CREB1/3: cAMP response element binding protein 1/3; DCN: Decorin; Esr1: Estrogen receptor 1; ETS1: v-ets erythroblastosis virus E26 oncogene homolog 1; FOXO1A: Forkhead headbox O1A; hESC: Human endometrial stromal cell; HOXA10: Homeobox A10; IGFBP-1: Insulin-like growth factor binding protein-1; IL-1: Interleukin-1; KLF12: Krüppel-like factor 12; miR-181a: microRNA-181a; MPA: Medroxyprogesterone acetate; PR: Progesterone receptor; PRL: Prolactin; RIF: Repeated implantation failure; TNF-α: Tumor necrosis factor-α;3 ′UTR: 3′ untranslated region; 8-Br-cAMP: 8-bromoadenosine-cAMP. Competing interests The authors declare that they have no competing interests. Authors’ contributions QZ, HZ, and YJ carried out the molecular genetics studies. XZ contributed to the isolation of hESC. QZ and GY drafted the manuscript. BX, ZD, LD, and HS participated in the design of the study and performed the statistical analysis. GY and YH conceived of the study, and participated in its design. All authors read and approved the final manuscript. Acknowledgments This work was supported by the National Natural Science Foundation of China (81370683, 81170570, 81370724, and 81070492), a special grant for principal investigators from the Health Department of Jiangsu Province (LJ201102 and RC2011005) and the program for the six top talents of Jiangsu Province, PR China (No. 2012-WSN-005). Author details 1Reproductive Medicine Center, Department of Obstetrics and Gynecology, Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing 210008, Jiangsu, China. 2Reproductive Medicine Center, Drum Tower Clinic Medical College of Nanjing Medical University, Nanjing 210029, Jiangsu, China. Received: 11 November 2014 Accepted: 14 March 2015

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Nat Commun. 2014;5:2977. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Zhang et al. Reproductive Biology and Endocrinology (2015) 13:23 Page 9 of 9