Elevated Tristetraprolin Impairs Trophoblast Invasion in Women with Recurrent Miscarriage by Destabilization of HOTAIR.

Tristetraprolin (TTP) regulates the stability of multiple targets that have important biological roles. However, the role of TTP in trophoblasts at the maternal-fetal interface remains poorly understood. We demonstrated that TTP was upregulated in placental trophoblasts from patients with recurrent miscarriages (RMs). Immunofluorescence and immunoblotting analyses indicated that TTP was redistributed from the nucleus to the cytoplasm in trophoblasts from patients with RMs. Trophoblast invasion and proliferation was repressed by TTP overexpression and was enhanced by TTP knockdown. Interestingly, TTP knockdown promoted trophoblast invasion in an ex vivo explant culture model. Furthermore, TTP overexpression in trophoblasts significantly inhibited the expression of the long non-coding RNA (lncRNA) HOTAIR. TTP was found to regulate HOTAIR expression by a posttranscriptional mechanism. To RNA immunoprecipitation (RIP) and RNA-protein, pull-down identified TTP as a specific binding partner that decreased the half-life of HOTAIR and lowered steady-state HOTAIR expression levels, indicating a novel posttranscriptional regulatory mechanism. Our findings identify a novel function for TTP in lncRNA regulation and provide important insights into the regulation of lncRNA expression. This study reveals a new pathway governing the regulation of TTP/HOTAIR in trophoblast cell invasion during early pregnancy.

Introduction

Successful embryo implantation depends on fertilization, trophoblast development, and proper maternal-fetal cross-talk and immune regulation. Two major trophoblast cell lineages have been identified during the early stages of human placental development: syncytiotrophoblast (STB) and cytotrophoblast (CTB).2, 3 Cytotrophoblasts within the villous placenta differentiate into extravillous trophoblasts (EVTs), which have an invasive phenotype. EVTs invade the decidua and maternal spiral arteries from the first trimester until 24 weeks of gestation. This invasion is critical to maternal spiral artery remodeling and the promotion of maternal placental blood flow, necessary to establish effective maternal-fetal exchange.5, 6 Impairment of EVT invasion predisposes a pregnancy to uteroplacental insufficiency and a significantly increased risk of pre-eclampsia, fetal growth restriction (FGR), and early recurrent miscarriage (RM).7, 8 RM affects approximately 1%–3% of females during their reproductive years and usually is defined as three or more consecutive spontaneous abortions before 20 weeks of gestation. To date, RM remains a frustrating challenge for clinicians and a distressing problem for patients. Thus, comprehensive molecular studies are required to better understand the causes of RM and to identify potential biomarkers that could be potential therapeutic targets.

Trophoblast invasion is temporally and spatially regulated by autocrine and paracrine interactions from trophoblastic and uterine factors at the maternal-fetal interface. This regulation guarantees that invasion of trophoblast cells only occurs during the early stages of gestation and is restricted to the whole maternal endometrium and the upper third of the myometrium. A complex network of cell types, mediators, and signaling pathways regulating trophoblast invasiveness has been described, and a link has been established between regulation of long non-coding RNAs (lncRNAs) and placental development. The lncRNAs MEG3 and MALAT1 have been shown to contribute to the behavior of trophoblast cells in pre-eclampsia. Recently, we showed that HOTAIR expression was significantly lower in trophoblasts from patients with RM compared to health controls. We also found that HOTAIR regulates trophoblast migration and invasion, thereby contributing to the pathology of RM and/or pre-eclampsia, suggesting HOTAIR to be a potential therapeutic target for RM. This indicates that HOTAIR acts as an important non-coding RNA (ncRNA); however, the regulatory mechanisms controlling HOTAIR stability are largely unknown.

Tristetraprolin (TTP) is a tandem zinc-finger mRNA binding protein that binds to adenylate-uridylate-rich elements (AREs) in the 3′ UTRs of specific mRNAs. The 3′ UTRs of these specific mRNAs contain the consensus sequence of UUAUUUAUU; physiologically relevant confirmed target transcripts often contain multiple consensus binding sites in close proximity to each other. TTP and the related proteins ZFP36L1, ZFP36L2, and ZFP36L3 consist of two conserved CCCH zinc fingers and have similarly sized but divergent N- and C-terminal regions. TTP was first identified as an immediate early-response gene in cells stimulated with insulin, serum, or lipopolysaccharide (LPS). TTP mRNA is widely distributed among tissue types, with high levels of mRNA expressed in spleen, lymph nodes, and thymus.20, 21 TTP is a nucleo-cytoplasmic shuttling protein that is localized predominantly to the nucleus of cells in their basal state but becomes localized to the cytoplasm upon stress stimulus. Mice deficient in TTP exhibit aberrant inflammatory phenotypes, mostly explained by deregulation of the mRNA stability and biosynthesis of cytokines. The interaction of TTP with AREs in the 3′ UTR of targeted mRNAs, such as tumor necrosis factor alpha (TNF-α), promotes mRNA degradation. TTP mRNA and protein levels are increased in the reproductive tract of mice during the estrous cycle. In addition, the TTP family member ZFP36L1 may be crucial to female fertility and embryonic development, as disrupted ZFP36L1 resulted in infertility in mice. Recently, we reported TTP expression is increased in trophoblasts from patients with RM compared to healthy controls. Moreover, Kasra Khalaj et al. showed that TTP protein was downregulated in an LPS-induced mouse pregnancy loss model, and its targets TNF-α and interleukin-6 (IL-6) were upregulated.

In this study, we report that RM patients have significantly higher expression of TTP in chorionic villous tissue compared with age-matched normal controls; this is consistent with the finding that TTP overexpression inhibits trophoblast invasion and migration. Using RT2 lncRNA PCR arrays, we have identified the impact of TTP expression on expression profiles of lncRNA in trophoblasts. Furthermore, we established the relationship between the expression of HOTAIR and TTP expression in the pathogenesis of RM.

Results

TTP Expression Is Overexpressed in Trophoblasts from Patients with RM

Our previous gene expression microarray results showed that expression of TTP was higher in trophoblasts from patients with RM group (NCBI: GSE76862). Here, we further evaluated TTP expression using real-time qPCR and western blot analysis of first-trimester chorionic villi tissue to explore whether TTP is involved in the pathogenesis of RM. TTP expression was significantly upregulated in villous tissue of patients with RM (Figures 1A and 1B). Immunohistochemical analysis of paraffin-embedded first-trimester chorionic villous tissues was performed to investigate the localization of TTP in chorionic villous tissue. Expression of TTP in normal chorionic villous tissue from healthy controls (HCs) mainly exhibited nuclear staining in cytotrophoblasts (CTBs), but not in syncytiotrophoblasts (STBs). A stronger positive signal for TTP was detected in chorionic villous tissue from the RM group compared to the HC group (Figures 1C and 1D). Furthermore, fluorescence staining using an anti-TTP antibody revealed that TTP was mainly expressed in nucleus of CTBs from HCs sample, and TTP expression was detected in the nucleus and cytoplasm of CTBs from RM samples (Figure 1E). These findings were confirmed by western blot and qRT-PCR analysis, which showed that TTP was expressed at a higher level in primary trophoblast of RM patients than that of HCs (Figures 1F and 1G). Moreover, nuclear-cytoplasmic fractionation and western blot analysis showed that TTP was distributed mainly in the nuclei of primary trophoblasts isolated from HCs sample, whereas significant amounts of TTP could also be detected in the cytoplasm and nuclei of primary trophoblasts isolated from RM patients (Figures 1H and 1I). Together, these results indicate that TTP expression is increased in trophoblasts in RM patients and suggest that this increase may be correlated with trophoblast proliferation and invasion.

Figure 1

TTP Is Upregulated in First-Trimester Placental Cytotrophoblasts in RM Patients

(A and B) Tristetraprolin (TTP) expression in first-trimester human villi tissues from patients with recurrent miscarriage (RM) or in healthy controls (HCs) was determined by qRT-PCR (A) and western blotting (B) analysis. **p < 0.01 compared with HCs. (C) Single staining of maternal villi (cytotrophoblasts and syncytiotrophoblasts) using anti-IgG (rabbit) or anti-TTP antibody, visualized with labeled streptavidin biotin horseradish peroxidase (HRP) kit is shown. (D) Sections were counterstained with hematoxylin, and positive cells were quantified using ImagePro-plus 6.0 software (n = 16); right scale bar represents 100 μm; left scale bar represents 25 μm. (E) Immunofluorescence of TTP in primary trophoblasts from first-trimester decidual tissue (8–10 weeks of gestation) of RM patients (n = 15) and healthy controls (n = 15) is shown. Fluorescence signals specific to anti-IgG or anti-TTP antibodies appear green, and the DAPI-stained nuclei appear blue; scale bar represents 10 μm. *p < 0.05 compared with HCs. (F and G) Primary trophoblasts were isolated from first-trimester villi tissue of RM patients and healthy controls; levels of TTP expression in primary trophoblasts were detected by qRT-PCR (F) and western blotting (G). *p < 0.05 compared with HCs. (H and I) Primary trophoblasts were isolated from first-trimester villi tissue of RM patients and healthy controls, and levels of TTP expression were determined in the cytoplasm (H) and nucleus (I) using immunoblotting.

TTP Decreases Proliferation and Invasion of Trophoblasts In Vitro

We next investigated whether TTP is involved in the regulation of trophoblast proliferation and migration. The HTR-8/SVneo (HTR-8) cell line, a first-trimester human extravillous cytotrophoblast-derived cell line, was transfected with small interfering TTP (siTTP) oligonucleotides or with the TTP-expressing vector. TTP expression decreased after siTTP transfection and was upregulated after transfection with the TTP expression vector (Figure 2A). Overexpression of TTP resulted in a significant decrease in the proliferation of HTR-8 cells, as indicated by CCK-8 analysis, whereas knockdown of TTP increased HTR-8 proliferation (Figure 2B). Furthermore, wound healing assays and Matrigel invasion assays revealed that overexpression of TTP significantly decreased the migratory and invasive ability of HTR-8 cells, whereas knockdown of TTP significantly promoted migration and invasion (Figures 2C–2G). These results suggest that TTP plays a key role in trophoblast proliferation, migration, and invasion.

Figure 2

TTP Inhibits Trophoblast Proliferation and Invasive Ability In Vitro

(A) Western blot analysis of TTP expression in HTR-8 cells transfected with siCtrl, siTTP#1, siTTP#2, siTTP#3, control vector, or TTP-overexpressing vector after 48 hr. (B) HTR-8 cells were transfected with these four vectors and incubated for 48 hr. Cell proliferation was measured after 48 hr using the CCK-8 assay. *p < 0.05 compared with siCtrl or control vector. (C) Overexpression of TTP in HTR-8 cells resulted in decreased wound closure ability compared to the cells transfected with vector control. TTP knockdown drastically increased the rate of wound closure in comparison to the siCtrl cell line (magnification ×100). (D–G) Overexpression of TTP in HTR-8 cells significantly reduced cell invasion compared to the vector control cell line (left) (D). The cells’ ability to invade was assessed using ImagePro 6.0 software (E and G). Knockdown of TTP promoted cell invasion compared to the scrambled control cell line (F). The scale bars represent 50 μM. *p < 0.05 versus siCtrl or control vector.

TTP Reduces the Migration of EVTs in an Extravillous Explant Culture Model

To further elucidate the role of TTP in trophoblast invasion and migration in vitro, placental villi with EVT cell columns were explanted from the RM and control groups (6–10 weeks of gestation) and cultured on Matrigel-coated dishes. The distance of outgrowth on the Matrigel surface was measured at 24 hr and 72 hr to assess EVT cell migration. EVT outgrowth was visible 24 hr post-explant culture. No significant difference was observed between the control and RM groups at 24 hr. After 72 hr of in vitro culture, the RM explants exhibited reduced migration compared to the control explants (Figures 3A and 3B). Further, whole-mount immunofluorescence analysis showed that the TTP expression was significantly increased in the RM EVT explants compared with the control explants (Figure 3C). To further clarify the regulation of trophoblast invasion by TTP, explants freshly obtained from the RM villous tissue samples were cultured in 24-well dishes for 24 hr and then treated with siCtrl and siTTP oligonucleotides. Explants treated with siTTP migrated significantly farther than the siCtrl-treated explants (Figures 3D and 3E). Together, these results are consistent with our finding that TTP inhibits trophoblast invasion, supporting the notion that TTP might be the key mediator of RM.

Figure 3

TTP Regulates Trophoblast Outgrowth in Extravillous Explant Cultures

(A) Extravillous explants were obtained from HCs and RM patients at 6–8 weeks of gestation and cultured on Matrigel; scale bar represents 250 μM. (B) Statistical analysis of the migration distance of villous tips (%) is shown. Data are presented as means ± SD of three independent experiments. *p < 0.05 compared with HCs. (C) The boxed areas in (A) are enlarged and shown in (C). At 72 hr, the extravillous explants were performed immunofluorescence staining using anti-TTP antibodies. The results showed an increase in TTP protein levels in the RM group compared to the HC group. Green fluorescence signals indicate bound anti-TTP antibodies, CK7 staining is visualized as red, and DAPI-stained nuclei are blue; scale bar represents 25 μM. (D) Extravillous explants from RM patients were maintained in culture on Matrigel. Serial pictures of the explants incubated with siTTP or siCtrl were taken under a light microscope after 24 hr and 72 hr of culture in vitro; scale bar represents 250 μM. (E) Statistical assay of the migration distance of villous tips (%) was measured using ImagePro J 6.0. Data are presented as means ± SD of three independent experiments. *p < 0.05 compared with siCtrl.

HOTAIR Is a Downstream Target of TTP in Trophoblasts

To identify potential targets of TTP, primary trophoblasts obtained from first-trimester placental tissue of human subjects were transfected with the control or TTP expression vectors. Next, expression profiles of 84 lncRNAs were analyzed on the RT2 lncRNA PCR array and compared between primary human trophoblasts transfected with the control vector or the TTP expression plasmid. We identified 8 lncRNAs that were upregulated and 11 lncRNAs that were downregulated after TTP overexpression in primary trophoblasts (Figure 4A). Of these, the expression of HOTAIR was decreased significantly in trophoblasts after TTP overexpression. Moreover, overexpression of TTP also decreased the expression of HOTAIR in HTR-8 cells, and knockdown of TTP significantly promoted expression of HOTAIR (Figures 4B and 4C). Overexpression of HOTAIR was able to rescue the reduction of HOTAIR induced by TTP overexpression (Figure 4D). Additionally, overexpression of HOTAIR reversed the reduction in trophoblast invasion caused by overexpression of TTP (Figures 4E–4H). Taken together, these results indicate that TTP regulates trophoblast migration via downregulation of HOTAIR expression.

Figure 4

HOTAIR Is a Downstream Target of TTP in Trophoblasts

(A) Real-time PCR was performed to determine the levels of long non-coding RNAs (lncRNAs) in trophoblasts transfected with control vector or TTP overexpression vector for 48 hr. (B and C) qRT-PCR analysis of HOTAIR expression in HTR-8 cells transfected with control vector, TTP expression vector (B), and shCtrl, shTTP#2, or shTTP#3 (C) as indicated. *p < 0.05 compared with siCtrl or vector. (D) qRT-PCR analysis of HOTAIR expression in HTR-8 cells that were transfected with vector or TTP overexpression plasmid or with TTP + HOTAIR overexpression plasmid is shown. *p < 0.05 compared with vector or TTP. (E–G) HTR-8 cells (E and F) and primary trophoblasts (G and H) were transfected with vector or TTP overexpression plasmid or with TTP + HOTAIR overexpression plasmid for 24 hr. The invasive ability of the cells was assessed by crystal violet staining; scale bars represent 50 μM. *p < 0.05 compared with vector or TTP.

TTP Specifically Regulates HOTAIR Expression and Stability in Trophoblasts

Four TTP binding sites were predicted in adenosine and uridine (AU)-rich elements of HOTAIR, based on the AUUUA consensus sequence. These four binding sites were selected for further luciferase reporter screening (Figure 5A). The full sequence of HOTAIR was obtained by PCR amplification of human trophoblast cDNA. This fragment was subcloned into the psiCheck 2.0 luciferase reporter vector (Figure 5B). Reporter plasmids were transfected into HTR-8 cell in conjunction with the control vector or TTP expression plasmid. After 24 hr, the reporter activity was measured by a dual-luciferase assay. The deletion variants of the HOTAIR region yielded different basal levels of expression of the luciferase gene. Two variants, ARE 2-4 and ARE 3-4, almost completely rescued the luciferase activity suppressed by TTP, indicating that the reporter activity suppression conferred by TTP was mediated by the ARE 2-4 and ARE 3-4 sites (Figure 5C). To further elucidate molecular mechanisms of TTP regulation of HOTAIR expression, hnRNPA1, an unrelated RNA binding protein, was included as a control for specificity. HTR-8 cells were transfected with vector or with hnRNPA1 expression plasmid. HnRNPA1 expression increased after hnRNPA1 transfection, but TTP expression was not changed after transfection of the hnRNPA1-expression vector (Figure 5D). To further distinguish between a transcriptional and a posttranscriptional regulatory mechanism, we assessed the mRNA half-life of HOTAIR in the TTP- or hnRNPA1-expressing trophoblasts using an actinomycin D chase experiment and a one-phase exponential decay model with GAPDH mRNA normalization. This experiment revealed a strong impact of TTP on HOTAIR RNA stability. Although the half-life of HOTAIR was between 7.7 hr after control vector and hnRNP A1 plasmid transfection, the half-life was almost two-fold shorter when cells were transfected with TTP vector (4.4 hr; Figures 5E and 5F). These results suggest that TTP plays a role in mediating stability of HOTAIR RNA.

Figure 5

Identification of TTP as an Interaction Partner with HOTAIR in Trophoblasts

(A) The diagram illustrates the TTP binding site in the primary transcript region of HOTAIR on the human chromosome. The human DNA sequences of putative TTP binding sites are labeled as ARE 1-4, and the core element (AUUUA) for TTP binding is indicated with red. (B) A schema for the constructs and co-transfection experiments is shown. (C) A fragment of the 3′ UTR of HOTAIR was cloned in a psiCheck 2 vector, downstream of the Renilla luciferase gene. These constructs where co-transfected with the TTP overexpression vector and with an empty vector into HTR-8 cells. Cells were harvested after 48 hr; Renilla luciferase activity was measured and normalized to firefly luciferase. Results are represented as the mean of three independent experiments. *p < 0.05 compared with vector. (D) Western blot analysis of TTP and hnRNPA1 expression in HTR-8 cells transfected with control vector or with hnRNPA1-overexpressing vector after 48 hr is shown. (E and F) HOTAIR stability analysis in HTR-8 cells after actinomycin D (5 μg/mL) treatment is shown. Cells were transfected with vector, TTP- (E), or hnRNPA1- (F) overexpression plasmid for 36 hr; a time course for RNA stability was initiated by adding the RNA polymerase II inhibitor (actinomycin D). Cells were harvested at the indicated time points. Expression levels were normalized to “0 hr,” and GAPDH was used as the reference gene. Results are shown as the mean of at least three independent experiments. *p < 0.05 compared with vector. (G) Analysis of co-purified RNA and respective enrichment as determined by qRT-PCR after anti-TTP or anti-hnRNPA1 immunoprecipitation validating specific binding of HOTAIR RNA to TTP is shown. All immunoprecipitation experiments were done in biological replicates (n = 3). (H) Validation of specific binding of TTP to biotinylated HOTAIR in HTR-8 cell by western blotting is shown.

We further performed RNA immunoprecipitation assays to further investigate the role of TTP in regulation of HOTAIR expression. TTP or hnRNPA1 (negative control) were transiently overexpressed in HTR-8 cells and immunoprecipitated with either anti-TTP or anti-hnRNPA1 antibodies. After isolation of the co-purifying RNA, the enrichment of selected transcripts was measured by qRT-PCR, and we confirmed the specific enrichment of HOTAIR (Figure 5G). Furthermore, we aimed to identify HOTAIR-interacting proteins as potential regulators using an RNA affinity purification approach. Cytoplasmic extracts prepared from HTR-8 cells were incubated with a 1,200-nt-long HOTAIR RNA that was transcribed in vitro and biotinylated. An RNA molecule of the same length but unrelated in sequence was used as a negative control. Proteins associated with HOTAIR or the control RNA were eluted and separated on a polyacrylamide gel and the level of TTP and hnRNP A1 expression detected with western blotting (Figure 5H). These results indicated that TTP, not hnRNPA1, regulates HOTAIR posttranscriptionally.

TTP Is Overexpressed in RM Disease and Correlates Negatively with HOTAIR Expression

We further examined the expression levels of HOTAIR and TTP mRNA in trophoblasts from patients with one miscarriage, two miscarriages, RM disease, and healthy controls. Consistently, we found a significantly higher level of TTP mRNA and a significantly lower level of HOTAIR mRNA in the trophoblasts from the RM group (Figures 6A and 6B). Linear correlation analysis showed that the HOTAIR level was negatively correlated with the TTP level in villous tissue (Figure 6C). Thus, we propose a model in which TTP binds to and negatively regulates HOTAIR expression in trophoblast cells, thereby mediating RM disorder (Figure 6D).

Figure 6

Expression of TTP Is Negatively Correlated with Expression of HOTAIR in Villous Tissue from Patients with RM

(A and B) The HOTAIR (A) and TTP mRNA (B) expression levels in the villous tissue of patients with one (n = 17) miscarriage or two (n = 15) miscarriages, RM (n = 27), and HCs (n = 25) were determined by qRT-PCR. **p < 0.01 compared with HCs. (C) HOTAIR and TTP mRNA expression levels were measured in the villous tissue of patients with RM and healthy controls (n = 38) using qRT-PCR; HOTAIR expression correlated negatively with TTP mRNA expression levels. (D) Diagrammatic representation of the study results. (Above) In normal pregnancy, lower expression of TTP promotes expression of the HOTAIR gene, which maintains trophoblasts in a state of normal invasion and proliferation. (Below) In RM, this circuit becomes dysregulated due to increased expression of TTP, which constitutively represses HOTAIR. The lower expression of the HOTAIR inhibits trophoblast invasion and proliferation, thereby leading to RM.

Discussion

Our study elucidates the biological role of TTP in trophoblast function and implicates TTP in the pathogenesis of RM (Figure 6D). Overexpression of TTP in vitro inhibited cell proliferation and invasion of the first-trimester human extravillous trophoblast-derived HTR-8 cell line and in primary trophoblasts. In addition, this study describes a comprehensive mechanism for TTP in the pathogenesis of RM, whereby TTP binds to the AREs of the lncRNA HOTAIR to regulate HOTAIR expression. Our results suggest that the TTP/HOTAIR pathway may serve as a novel therapeutic target for RM disease, which is characterized by failed embryonic implantation and insufficient trophoblast invasion. The findings from this research have clinical implications for patients with RM as well as for other pregnancy-related disorders associated with elevated TTP and/or shallow trophoblast invasion. Nevertheless, the elucidation of the mechanisms underlying upregulation of TTP in RM placental tissue and the relationship between TTP and the onset of RM remains to be investigated.

Posttranscriptional regulation through specific RBPs is emerging as a critical regulatory level in nearly all biological processes. TTP is a kind of RBP, and it plays an important role in stabilizing and/or modulating the translation of many of its target mRNAs, including the RNAs encoding c-fos, vascular endothelial growth factor (VEGF), MKP-1, TNF-α, Bcl-2, Mcl-1, and COX-2. In this study, we found that the level of TTP is relatively higher in the placental tissue from RM patients as compared with matched healthy controls, suggesting that TTP is disrupted under pathological conditions. Moreover, we have previously reported that HOTAIR regulates MMP-2 expression and promotes trophoblast invasion. Here, we report that TTP can act as an RNA binding protein that contributes to destabilization of HOTAIR in human trophoblasts. These results suggest the TTP/HOTAIR pathway could play an important role in trophoblast invasion.

To understand the mechanisms underlying RM, a large number of gene-profiling studies have been conducted. These studies mainly focused on the role of protein-coding genes and rarely included long, non-coding transcripts. It was found that HOTAIR, which exerted regulatory transcription activity, could bind PRC2 and the LSD1/CoREST/REST complex and direct it to the specific target gene, causing H3K27 methylation and H3K4 demethylation and gene silencing.31, 32, 33, 34 However, surprisingly little is known about the general role of HOTAIR decay in the context of RM. Although factors such as microRNAs (miRNAs) and AU-rich element binding proteins are known to specifically target mRNAs for degradation, we are still far from a comprehensive understanding of the network that controls the stability of individual lncRNAs. Here, we demonstrate through gain- and loss-of-function analyses that HOTAIR expression during trophoblast invasion is TTP dependent, indicating that TTP is an important regulator of HOTAIR in trophoblasts. We further used luciferase reporter assays, RIP, and RNA-protein-interaction analyses to demonstrate that TTP acts as a trans-acting factor that represses HOTAIR stability and expression. Therefore, TTP might be involved in the progression of RM via modulating HOTAIR expression.

In conclusion, our research demonstrates that TTP acts as a key regulator of HOTAIR expression and trophoblast function. Because (1) TTP is upregulated while HOTAIR is downregulated in RM and (2) HOTAIR is a potent regulator of trophoblast proliferation, migration, and invasion—the processes deregulated in RM patients, we propose that TTP may be a useful therapeutic target for the treatment of RM.

Materials and Methods

Patient Characteristics

Between December 2016 and April 2017, 28 patients with RM (23–35 years old; mean age, 28.4 ± 6.6 years), 17 patients with one miscarriage between 24 and 35 years of age (mean age, 30.1 ± 3.9 years), and 15 patients with two miscarriages between 22 and 34 years of age (mean age, 27.6 ± 4.2 years) who had been treated at the Department of Obstetrics and Gynecology in the International Peace Maternity and Child Health Hospital, China Welfare Institute, Shanghai Jiao Tong University School of Medicine, China were recruited to this study. Patients with the following features were excluded: (1) presence of uterine abnormalities or cervical incompetence on pelvic examination and ultrasound; (2) abnormal karyotype analysis of the parents or abortus; (3) presence of autoimmune abnormality, luteal phase defects, hyperprolactinemia, or hyperandrogenemia, as indicated by comprehensive hormonal status assessment; and (4) symptoms of endocrine or metabolic diseases (e.g., diabetes, hyperthyroidism, and hypothyroidism).

Additionally, 29 women aged 22–35 years (mean age, 27.9 ± 7.1 years) with normal early pregnancies were recruited as healthy controls. All healthy controls had previous pregnancies and lacked any history of spontaneous abortion, preterm labor, or pre-eclampsia. All women recruited to the control group had undergone artificial abortions to terminate unwanted pregnancies at 6–10 weeks of gestation; villous tissue samples were collected from these patients and stored in liquid nitrogen until analysis. The study protocol was approved by the Medical Ethics Committee of the International Peace Maternity and Child Health Hospital of China Welfare Institute, Shanghai. Written informed consents were obtained from all the participants before enrollment.

Cell Culture

Primary trophoblasts were isolated by trypsin-DNase I digestion and discontinuous Percoll gradient centrifugation from pooled villous tissue samples obtained from 3–5 patients, as previously described. The isolated trophoblast cell cultures had a purity of approximately 95%, as determined by flow cytometry for cytokeratin-7-positive, human leukocyte antigen (HLA)-G-positive, and vimentin-negative cells (data not shown). Purified trophoblasts were seeded in the wells of 12-well plates at a concentration of 6 × 105 cells/mL and cultured in DMEM/F12 plus 10% fetal bovine serum (FBS) (Gibco).

The HTR-8/SVneo cell line (HTR-8), is derived from human invasive EVTs and was a kind gift from Dr. PK Lala (University of Western Ontario, London, ON, Canada). HTR-8 cells were cultured in RPMI medium supplemented with 10% FBS and antibiotics (penicillin and streptomycin).

Overexpression of TTP and HOTAIR

The LZRS-HOTAIR plasmid was a gift from Howard Chang (Addgene plasmid no. 26110). The pFRT-TODestFLAGHAhTTP plasmid was a gift from Thomas Tuschl (Addgene plasmid no. 48738). These plasmids and the control vector were purified using an Endofree Plasmid kit (QIAGEN) and transfected into cells using a jetPRIME kit (Polyplus Transfection).

Knockdown of TTP

TTP knockdown was performed using specific small interfering RNA (siRNA) specific for endoribonuclease-prepared siRNA TTP (esiTTP) (Sigma) and transfected into cells at a final concentration of 100 ng/mL using Oligofectamine reagent (Invitrogen). Lentiviruses carrying short hairpin RNAs (shRNAs) targeting human TTP were purchased from GeneChem (cat. GIEL2481085466; Shanghai, China) and transduced into primary trophoblast cells and HTR-8 cells according to the manufacturer’s instructions.

Explant Culture

Small 2- or 3-mm tissue sections were obtained from the tips of first-trimester human placental villi (6–10 weeks), dissected, and explanted in 24-well culture dishes pre-coated with phenol red-free Matrigel substrate (Corning). Coated inserts were placed into 24-well culture dishes (Costar, Cambridge, MA). Villi explants were cultured in DMEM/F12 media containing 10% FBS. Placental villi that successfully anchored on Matrigel matrix and initiated outgrowth were used for the subsequent experiments and are referred to as 24 hr samples. EVT sprouting and migration from the distal end of the villous tips were recorded daily for up to 3 days. The extent of migration was measured via ImageJ Pro 6.0 software. Extravillous explants from RM patients were incubated with TTP-specific siRNA or control siRNA with 250 ng/mL, and images were obtained after 24 hr and 72 hr of in vitro culture under a light microscope. Ten explants per experiment were analyzed from both the TTP siRNA and control groups.

Detection of lncRNAs in Trophoblasts

Total RNA was extracted from primary trophoblasts using TRIzol reagent, according to the manufacturer’s instructions. cDNA was synthesized from the extracted RNA using an RT2 PreAMP cDNA Synthesis Kit (QIAGEN, Germany), according to the manufacturer’s instructions. cDNA samples were then processed for analysis on the Human RT2 lncRNA PCR Array Human Cancer Pathway Finder (cat no. LAHS-002Z; QIAGEN, Duesseldorf, Germany), which contains 84 lncRNA genes that are related to Human Cancer Pathway; five housekeeping genes were used as controls. Statistical analysis was evaluated using web-based RT2 Profiler PCR Array Data Analysis.

RNA-Binding Protein Immunoprecipitation

RIP was performed using the EZMagna RIP Kit (Millipore, USA), according to manufacturer’s protocols. Briefly, HTR-8 cells were harvested with ice-cold PBS and were then lysed by RIP lysis buffer. Then RIPAb+ TTP (Millipore, USA), RIPAb+ heterogenous nuclear ribonucleoprotein A1 (hnRNPA1), and nonspecific control rabbit immunoglobulin G (IgG) antibodies were used for the immunoprecipitations. RIP lysates and antibody bound to magnetic beads were incubated together overnight at 4°C with rotation. Afterward, proteins in the immunoprecipitates were digested with proteinase K. Bound RNAs were purified from the supernatants, reverse transcribed using PrimeScript RT Reagent Kit, and evaluated by quantitative analysis. Relative RNA expression was calculated using the 2−ΔCt method and normalized against input values. The primers used for PCR were as follows: HOTAIR primer for RIP no. 1 F: 5′-AGAGAGAGGGAGCCCAGAGTTA-3′, R: 5′-CGTGGCATTTCTGGTCTTGTA-3′; HOTAIR primer for RIP no. 2 F: 5′-TTAGTTGCTAAGGAAAGATCCAAAT-3′, R: 5′-AGGCTTTCCTATAACCCAAGCT-3′.

In Vitro Transcription

The sense strands of full-length human non-coding RNA HOTAIR underwent in vitro transcription using the Megascript T7 promoter kit (Life Technologies, Carlsbad, CA), according to the manufacturer’ protocol. Briefly, 1 μg linearized plasmid template was used, and reactions were incubated for 18 hr in the presence or absence of Biotin-16-uridine triphosphate (UTP) (GE Healthcare). The ratio between UTP and Biotin-16-UTP was 18:1. The reaction was stopped by addition of 1 μL Turbo-DNase. RNA was extracted with phenol-chloroform followed by ethanol precipitation. RNA integrity and size were evaluated using agarose gel electrophoresis.

RNA-Protein Pull-Down

For in vitro RNA pull-down, 5 μL cell lysates from HTR-8 cells were incubated with 1 μg biotin-16-UTP-labeled HOTAIR antisense in immunoprecipitation buffer for 30 min at 25°C. After addition of 5 μL MyOne Streptavidin T1 Dynabeads (Invitrogen), the mixture was incubated for a further 30 min and subjected to five wash cycles with 500 μL IPB buffer for 5 min each. After the final wash, magnetic beads were resuspended in 12 μL protein-loading buffer and RNA-bound proteins were separated by SDS-PAGE and detected with anti-TTP or anti-hnRNPA1 by western blot analysis.

Statistical Analysis

Data were analyzed using an independent sample t test for comparison between the two groups, and multi-group comparison was carried out by one-way ANOVA followed by Tukey’s post hoc test. Correlations were analyzed using the Spearman’s rank correlation test. Data are presented as mean ± SD. All p values are two-sided. A p value of < 0.05 was considered statistically significant. All statistical values were calculated using SPSS 22.0 (IBM Company, Chicago, USA).

Author Contributions

Conceptualization, Y.Z., Y.L., and W.-H.Z.; Performing Experiments, F.-J.T., X.-Y.H., J.W., X.L., X.-L.M., and F.W.; Data Analysis and Interpretation, F.-J.T., F.W., W.-H.Z., and X.-R.L.; Writing, F.-J.T., J.Z., W.-H.Z., and Y.L.; Funding Acquisition, F.-J.T., Y.Z., and Y.L. All authors read and approved the final version of the manuscript.

Conflicts of Interest

The authors have no conflicts of interest to declare.