Introduction

Decidualization, the transformation of endometrial stromal cells into specialized secretory decidual cells, is important for pregnancy by restraining trophoblast invasion and ensuring adequate homeostasis. Both excessive and inadequate invasion may lead to disastrous consequences1. Although protein-coding genes have been extensively characterized during human decidualization, whether long intergenic non-coding RNA (LincRNA) has a role in decidualization has not been studied. LincRNAs are more and more recognized as active molecules instead of “transcriptional noise”. Studies have revealed the involvements of LincRNAs in regulation of multiple biological processes2. However, the LincRNAs that participate in decidualization remain unclear.

The convergence of cAMP and progesterone signaling pathways is important for human decidualization3. cAMP sensitizes human endometrial stromal cells (HESCs) to progesterone via activation of the protein kinase A (PKA) signaling3. In human ciliary smooth muscle cells, long intergenic non-coding RNA 473 (LINC473) expression is obviously induced upon activation of the cAMP signaling pathway4. Because cAMP is a critical mediator of human decidualization, we hypothesized that cAMP-induced LINC473 expression might be important for human decidualization. However, the regulation and functional role of LINC473 during human decidualization remain unknown.

As a major mediator of IL-6 family signaling, STAT3 is important for mouse embryo implantation5,6,7. In humans, inhibition of STAT3 expression will impair decidualization5,8. However, the precise mechanism underlying the regulation of STAT3 on stromal decidualization remains unknown.

In this study, we identified a cAMP-induced expression pattern of LINC473 in the progress of decidualization. The cAMP-activated STAT3 plays a critical role in the induction of LINC473. We further provided evidence that knockdown of LINC473 attenuates the expressions of decidual markers prolactin (PRL) and insulin-like growth factor binding protein 1 (IGFBP1).

Results

Regulation of LINC473 expression by cAMP

As a remarkable property of LincRNAs is their low conservation9, we first did the evolutionary characteristic assay of LINC473. Based on our evolutionary characteristic analysis, LINC473 was identified as a primate-specific LincRNA (Fig. 1A).

Figure 1
figure 1

LINC473 is induced during decidualization.

(A) Simplified hylogenetic tree showing the evolutionary characteristics of LINC473 in primates. (B) LINC473 expression increases at different time points after decidualization. (C) PRL expression during decidualization. (D) The expression of IGFBP1 during decidualization. Each treatment was performed with at least three biological replicates. Error bars represent standard errors. *P < 0.05.

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Under in vitro decidualization, real-time PCR showed a strong induction of LINC473 after 1, 2, 4 and 6 days of decidualization, respectively (Fig. 1B). IGFBP1 and PRL, two decidual markers, were also strongly expressed (Fig. 1C,D). These results indicated that decidual stimulus was sufficient to induce and sustain LINC473 expression.

In our study, in vitro decidualization was induced with cAMP, progesterone and estrogen. To examine how one of these distinct factors regulates LINC473, HESCs were treated with cAMP, progesterone or estrogen, respectively. Real-time PCR analysis demonstrated that LINC473 expression increased dramatically upon cAMP treatment, but not by progesterone or estrogen treatment (Fig. 2A). The transcriptional levels of IGFBP1 and PRL, were also up-regulated by cAMP significantly (Fig. 2B,C). We then examined effects of time and dosage courses of cAMP on LINC473 expression. The cAMP regulation on LINC473 was rapid and dosage-dependent (Fig. 2D,E). Together, these results showed that the induction of LINC473 during decidualization was under the control of cAMP.

Figure 2
figure 2

LINC473 induction is under the control of cAMP.

(A) Relative expression level of LINC473 after stromal cells were treated with E2, P4 or cAMP for 24 h. (B) Effects of E2, P4 or cAMP on PRL expression. (C) Effects of E2, P4 or cAMP on IGFBP1 expression. (D) The time courses of cAMP-induced LINC473 expression. (E) Effects of cAMP of different concentrations on LINC473 expression. Each treatment was performed with at least three biological replicates. Error bars represent standard errors. *P < 0.05.

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LINC473 regulation by cAMP via induction of STAT3

To further confirm the cAMP regulation on LINC473 transcription, LINC473 promoter reporter was constructed for luciferase analysis. Because cAMP signaling is transduced exclusively by PKA10, H89, a selective PKA signaling inhibitor, was used to investigate whether PKA signaling pathway is involved in the induction of LINC473. The cAMP inductions on LINC473 expression and promoter activity were both attenuated by H89 (Fig. 3A, B). Together, these results showed that the induction of LINC473 during decidualization is under the control of cAMP-PKA pathway.

Figure 3
figure 3

cAMP regulates LINC473 via H89.

(A) HESCs were treated with cAMP for 6 h. H89 prevents the cAMP-induced increase of LINC473 expression level. (B) HESCs transfected with LINC473 promoter reporter plasmid were treated with cAMP for 24 h. The promoter activity of LINC473 is decreased with H89 treatment. (C) Luciferase activity of LINC473-promoter reporters with different lengths after HESCs were treated with cAMP for 24 h. Each treatment was performed with at least three biological replicates. Error bars represent standard errors. *P < 0.05.

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To identify the functional promoter region regulated by cAMP, different lengths of promoter truncations were constructed. The luciferase activities of −1246, −718 and −468 bp promoter reporters increased remarkably after cAMP treatment (Fig. 3C). However, there’s no obvious induction on luciferase activity for the −163 and −50 bp fragments. These results indicated that the importance of the −163 bp to −468 bp region to the effects of cAMP.

STAT3 activation involves induction of IL-11 during the early stage of decidualization

Evidences have showed that non-coding RNAs are directly regulated by transcription factors11. Therefore, we explored whether LINC473 is controlled by known decidualization-related transcription factors. Based on our in silico analysis of the −163 to −468 bp region relative to the LINC473 transcription start site (TSS), there was one binding site for STAT3 in this region (Fig. 4A). Because STAT3 is essential for human decidualization8, we assumed that STAT3 may be involved in the regulation of LINC473. We first monitored STAT3 expression upon cAMP treatment. The level of STAT3 mRNA showed no change in the early stage of cAMP treatment (data not shown). However, the level of phosphorylated STAT3 protein was upregulated by cAMP administration (Fig. 4B). To determine whether STAT3 is required for LINC473 induction by cAMP, S3I-201 (S3I), a selective inhibitor of STAT3 phosphorylation, was used to treat cells at the presence of cAMP. The assay on LINC473 promoter activity showed that S3I administration weakened the induction of LINC473 promoter activity by cAMP (Fig. 4C). The real-time PCR results displayed a similar pattern (Fig. 4D). To further confirm the function of STAT3, we mutated the STAT3 binding site and found that the mutation of STAT3 binding site attenuated the cAMP effects on LINC473 promoter reporter activity (Fig. 4E). These results indicated that STAT3 is required for cAMP regulation on LINC473.

Figure 4
figure 4

cAMP regulation on LINC473 via STAT3.

(A) Schematic representation of LINC473 promoter region showing the location of STAT3 binding. (B) cAMP induces the phosphorylation of STAT3 protein. (C) Effects of STAT3 inhibitor S3I on LINC473 promoter activity when HESCs were treated with cAMP. (D) Effects of STAT3 inhibitor on cAMP-induced LINC473 expression. (E) Mutation of STAT3 binding site attenuated the cAMP induction on LINC473 promoter activity. (F) Stimulation of IL-11 mRNA expression by cAMP. (G) LINC473 expression is induced by IL-11 treatment in HESCs. Each treatment was performed with at least three biological replicates. Error bars represent standard errors. *P < 0.05.

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To explore the molecular pathways through which STAT3 is activated, we searched the microarray data from cAMP-treated HESCs12. Based on their analysis, the expression level of IL-11, an IL-6 family member, was rapidly induced by cAMP. Our real-time PCR results also confirmed the induction of IL-11 by cAMP in HESCs (Fig. 4F). LINC473 mRNA expression was upregulated by IL-11 administration (Fig. 4G). These findings demonstrate that the phosphorylation of STAT3 may be involved in cAMP induction of LINC473.

PGE2 regulation on LINC473 expression

As cAMP production stimulated by PGE2 is important for decidualization, we examined whether PGE2 can regulate LINC473 expression. We performed dosage and time course experiments of PGE2 treatment on LINC473 expression. Real-time PCR results showed that PGE2 upregulated LINC473 expression (Fig. S1 A,B). The expression of IL-11 was also up-regulated by PGE2 (Fig. S1C). Western blot showed that the level of phosphorylated STAT3 protein was increased upon PGE2 treatment (Fig. S1D). PGE2 regulation on LINC473 was attenuated by H89 (Fig. S1E).

Functions of LINC473 during decidualization

To analyze the function of LINC473, LINC473 was knocked down by siRNA under in vitro decidualization. Real-time PCR showed that transfection of HESCs with LINC473 siRNA significantly reduced LINC473 expression (Fig. 5A). LINC473 knockdown also led to significant reductions in the mRNA levels of both PRL and IGFBP1 in decidualized HESCs (Fig. 5B,C). These results revealed an essential role of LINC473 in human decidualization. Additionally, the expressions PGR, FOXO1, HOXA10, HOXA11 and WNT4 were down-regulated upon LINC473 knockdown (Fig. 5D). These results indicated the importance of LINC473 during decidualization.

Figure 5
figure 5

Differentially expressed decidualization-related genes in HESCS transfected with LINC473 siRNA.

(A) LINC473 expression after silencing with siRNA. (B) Knockdown of LINC473 decreases PRL expression in decidualized HESCs. (C) Knockdown of LINC473 decreases IGFBP1 expression in decidualized HESCs. (D) Effects of LINC473 siRNA on the expression of decidualization-related genes. The expression levels of PGR, FOXO1, HOXA10, HOXA11, WNT4 and CEBPB were decreased by LINC473 knockdown. Each treatment was performed with at least three biological replicates. Error bars represent standard errors. *P < 0.05.

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Discussion

In contrast to the well-studied microRNAs, LincRNAs act through diverse mechanisms, as both positive and negative regulators of gene expression. Thousands of LincRNAs have been annotated in humans13, but the functions on the great majority of them remain unclear. A remarkable property of LincRNAs is their low interspecies conservation. Up to date, there are approximately 11,000 primate-specific LincRNAs and 2,500 conserved LincRNAs9. Our findings demonstrated that primate-specific LINC473 might play a critical role in human decidualization.

Several long non-coding RNAs have been reported in human uterus, including HOXA11 antisense14, SRA gene15, FGF-AS16 and EMX2OS17. LincRNA HOTAIR is reported to be important in the endometrial carcinogenesis18. However, the functional role of LincRNAs during the process of decidualization has not been reported.

There is overwhelming evidence that initiation of the decidual process requires cAMP signaling3. When HESCs were treated with progesterone, estrogen or cAMP, a significant LINC473 induction by cAMP was observed, but not by progesterone or estrogen, suggesting that cAMP might be the key inducing factor of LINC473 under human in vitro decidualization. PKA is the principle intracellular target for cAMP in mammalian cells19. In this study, PKA inhibitor H89 prevented the cAMP-dependent enhancement of LINC473. It has been reported that cAMP can induce human stromal cells to secrete IL-11 as decidualization progresses20. We showed that treatment of IL-11 can increase the LINC473 expression, suggesting that cAMP, at least partially, stimulates the expression of LINC473 via an IL-11-dependent pathway. As PGE2 has been considered as a physiological generator of cAMP21, we examined whether PGE2 can regulate LINC473 expression. The expression and secretion of IL-11 can be induced by PGE2 in HESCs20. It is possible that PGE2-stimulated cAMP induces the expression of IL-11, which drives the induction of LINC473.

Previous studies have identified LincRNAs that are directly regulated by transcription factors22. Transcription factor STAT3 has been identified as an critical regulator during human decidualization8. Our promoter analysis indicated that the cAMP-sensitive promoter region of LINC473 located from –163 to –468 bp contains STAT3 binding site. Here we report a unique finding that, besides protein-coding gene, STAT3 regulates the decidualization progress through LINC473.

LincRNAs could work either in cis or in trans. Their roles in regulating gene expression could be negative or positive23. To study the mechanism of LINC473 regulation, we first assumed the in cis regulation. Phosphodiesterase 10 (PDE10) gene, coding an enzyme hydrolyzing cAMP24, is the nearest gene in the LINC473-localized chromatin. However, no change of PDE10 expression was observed with LINC473 knockdown (data not shown), suggesting that no relationship exists between these two molecules.

In this study, we provided additional evidence of LINC473 regulation on several crucial decidual transcription factors and WNT4. Our results showed that LINC473 knockdown leads to a marked down-regulation of FOXO1, progesterone receptor (PGR), HOXA10, HOXA11 and WNT4 at the transcriptional level, but did not significantly affect the expression of CEBPB. Among these molecules, FOXO1 is important as a mediator of stromal cell decidualization, protection against oxidative damage and menstrual shedding25. Like LINC473, FOXO1 is also an induced target of PKA pathway26. Transcriptome assay reveals that over half of the FOXO1 target genes were regulated under decidualization26. During decidualization, FOXO1 is required for the PGR binding to its targets. The comparison of FOXO1 and PGR ChIP-seq data reveals over 75% co-occupancy26. Decreased FOXO1 is related to decidualization failure in women with endometriosis27. MiRNA regulation on FOXO1 has been reported during human decidualization28. Our results extend the finding that FOXO1 is also regulated by LincRNA during decidualization.

In summary, we have shown that LINC473 can significantly contribute to decidualization. However, the cellular function of LincRNAs remains enigmatic. In this regard, our data contribute to a growing body of literatures supporting the importance of LincRNA in cellular biology, especially during decidualization. As the potential of LincRNA to be a therapeutic target has been discussed recently29, the identification and functional analysis of LINC473 may add another layer of mechanism and foster novel markers or therapeutic options for clinical application, such as infertility and reasonable contraception.

Materials and Methods

Cell culture and in vitro induced decidualization

Human uterine stromal cells were isolated as previously described30. Human endometrial samples were collected from normally cycling women undergoing hysterectomy or endometrial biopsy with written informed consent. All human procedures were approved by the Institutional Committee on the Use of Human Subjects in Medical Research of Bailu Hospital (Xiamen, China). The methods were carried out in accordance with the approved guidelines by South China Agricultural University. Briefly, endometrial tissues were minced into small pieces and incubated in DMEM/F-12 containing 0.2% type I collagenase (Gibco) for 60 min at 37 °C. Single cells were dispersed mechanically by vibrating. The resultant single cell suspension was separated by successive filtrations through a 200 μm cell strainer. To induce decidualization, cells were treated with 10 nM E2 (Sigma), 0.5 mM db-cAMP (Sigma) and 1 μM medroxyprogesterone acetate (Sigma).

Sequence conservation analysis

Human LINC473 sequence was retrieved from Ensembl (http://www.ensembl.org/). The conserved sequences in other species were identified by PhastCons, UCSC (http://genome.ucsc.edu/). When more than 90% of human LINC473 was covered in the alignment, the segment was considered as present in other species. All identified sequences were aligned using ClustalW2 to construct the phylogenetic tree.

Real-time RT-PCR

Cells were harvested with TRIzol Reagent (Sigma) and RNA was extracted according to the manufacturer’s instructions. After digesting with RQ1 deoxyribonuclease I (Promega), total RNAs were reverse transcribed into cDNA with PrimeScript reverse transcriptase reagent kit (TaKaRa) and then real-time RT-PCR was performed using SYBR Premix Ex Taq kit (TaKaRa) on the Rotor-Gene Q system (BioRad). All reactions were run in triplicate. The ΔΔCt method was employed to determine relative changes of gene expression compared to GAPDH. Primer sequences used were listed in Table S1. Quantifications were performed with standard curves generated with pGEM-T plasmids containing specific cDNA amplicons.

Western blot

In brief, HESCs were lysed in lysis buffer (50 mM Tris-HCl, pH7.5, 150 mM NaCl, 1% Triton X-100 and 0.25% sodium deoxycholate). Protein concentration was quantified using the BCA kit (Applygen). Lysates were then resolved on a 10% SDS-PAGE gel and transferred onto PVDF membrane (Millipore). After blocking with 5% skim milk (Sangon), membranes were probed with the corresponding antibodies for phospho-STAT3 (Cell Signaling), total STAT3 (Cell Signaling) and Tubulin (Cell Signaling), respectively. Membranes were then incubated with the matched secondary antibodies conjugated with HRP (1:5000). Signals were detected with ECL kit (Pierce).

Plasmid Construction and Transfection

Different lengths of LINC473 promoter fragments were amplified using primers listed in Table S2 by PCR from human genomic DNA. The size of PCR products and their distance from TSS were also shown in Table S2. The amplified fragments were digested by restriction enzymes Mlu I and Hind III and inserted into the upstream of the start codon of luciferase gene in the pGL3-basic vector (Promega).

The mutation of STAT3 binding site in the promoter of LINC473 was generated with Quik ChangeTM Site-Directed Mutagenesis Kit (Stratagene, CA, USA). The primers used for the mutation were 5′-TCCGGCTGAACCCGCCCGAGCGCCCGCTC -3′and 5′-GAGCGGGCGCTCGGGCGGGTTCAGCCGGA -3′, in which the mutated sites were underlined. All the constructs were verified by sequencing.

Promoter reporter assay

Transfections were performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer. Six hours after reporter transfection, cells were treated with cAMP, H89 or S3I, respectively. Cell extracts were assayed for firefly and renilla luciferase activities (Promega E1910).

siRNA transfection

RNA interference was performed by using synthetic siRNA duplexes. siRNA oligonucleotides targeting LINC473 were designed and synthesized by Genepharma (Shanghai, China). HESCs were transfected with siRNA using Lipofectamine 2000 (Invitrogen) as manufacturer’s instructions. Cells were harvested using TRIzol (Invitrogen) for Real-time PCR or lysis buffer for Western blot assay.

Additional Information

How to cite this article: Liang, X.-H. et al. Non-coding RNA LINC00473 mediates decidualization of human endometrial stromal cells in response to cAMP signaling. Sci. Rep. 6, 22744; doi: 10.1038/srep22744 (2016).