Down-regulated lncRNA AGAP2-AS1 contributes to pre-eclampsia as a competing endogenous RNA for JDP2 by impairing trophoblastic phenotype.

Abstract Recently, growing evidence has shown that aberrant long non‐coding RNA (lncRNA) expression in conjunction with an impaired trophoblastic phenotype could implicate the pathological process of pre‐eclampsia (PE). However, only a small portion of lncRNAs has been characterized with regard to the function and molecular mechanisms involved in PE. There are still gaps in the available knowledge; as a result, there are currently only a few applicable treatments for PE in the context of lncRNA. Here, we found that lncRNA AGAP2‐AS1 is abnormally down‐regulated in severe PE placenta tissues. Using human trophoblasts, we established that AGAP2‐AS1 knockdown could inhibit trophoblasts proliferation and invasion and promote cell apoptosis. Further, we showed that overexpression of AGAP2‐AS1 substantially stimulated the development of the trophoblastic phenotype. Through high‐throughput sequencing analysis, we demonstrated that silencing of AGAP2‐AS1 favourably regulated various genes which are relevant to trophoblastic growth and invasion. Mechanistically, AGAP2‐AS1 promoted the suppressor protein, Jun dimerization protein 2 (JDP2), by sponging miR‐574‐5p. Resultantly, further impairment of the trophoblastic phenotype was achieved by way of inhibiting cell growth, apoptosis and invasion. We also determined that the expression of AGAP2‐AS1 could be mediated by FOXP1. Our results showed that the down‐regulated expression of lncRNA AGAP2‐AS1 might serve as a key suppressor in PE via inhibition of JDP2 at the post‐transcriptional level by competing for miR‐574; thus, this presents a novel therapeutic strategy for PE.


| INTRODUC TI ON
Pre-eclampsia (PE) is a distinct hypertensive pregnancy disorder that occurs in approximately 5-8% of females worldwide, and the incidence of PE has gradually increased. 1 Around 10% of all severe cases of PE cases occur before 34 weeks of gestation. Currently, PE is considered to be a multisystem syndrome of human gestation and is related to high maternal and infant morbidity and mortality rates. 2,3 Pregnancy predisposes females to a higher risk of pulmonary oedema, hepatic and renal failure, seizures, cerebral haemorrhage and even death. 4 Additionally, PE can impact the overall health and well-being of the foetus. Foetal growth restriction is a common complication associated with PE, and it is considered to occur as a result of chronic placental hypoperfusion. Placental abruption and premature separation of the placenta with the disruption of blood flow to the foetus are dangerous, yet uncommon, complications that occur in up to 3% of pregnancies affected by severe PE. 5 At present, the specific causes of PE in pregnancy remain unclear; moreover, no accurate method is available to prevent the development of PE.
Thus, there is an imperative need for the development of strategies that enable the safe delay of the delivery of babies to decrease the maternal and neonatal risks associated with premature birth.
Less than 2% of the mammalian genome includes protein-coding genes, about 90% of the genome accounts for lncRNAs 6 and the latter are transcribed rather than encoded proteins. Despite the lack of a general consensus regarding their biological characteristics, there is growing evidence that lncRNAs have a high degree of regulation and important functions in human diseases. Specifically, lncRNAs have emerged as key molecular players in the regulation of gene expression in different biological processes such as parental imprinting, stem cell pluripotency, cell growth, 7,8 and metastasis and apoptosis. 9,10 It is considered that these lncRNAs are regulated through effects such as miRNA sponging, chromatin remodelling and epigenetic modification. 11 It is interesting to note that recently, an increasing number of studies have validated the fact that lncRNAs might be associated with diverse human pregnancy-related disorders, especially PE. [12][13][14] AGAP2-AS1, a 1567 nt lncRNA located on chromosome 12q14.1, has been found to be abnormally expressed in multiple disease states, including cancer. Recent evidence shows that AGAP2-AS1 could epigenetically regulate MyD88 expression to promote chemoresistance in patients with breast cancer. 15 In addition, Li et al 16 reported that the elevated expression of AGAP2-AS1 regulates downstream target transcription in non-small-cell lung cancer by interacting with epigenetic proteins. Thus, in this study, we investigated the role of AGAP2-AS1 in the biological mechanism of PE as, at present, its role remains unclear.
In this report, through qRT-PCR in PE placenta tissues, we found that the RNA levels of AGAP2-AS1 were abnormally down-regulated, further suggesting that it may be connected to the pathogenesis of PE. Subsequently, we investigated the trophoblastic phenotype, including cell growth, apoptosis and invasion, after knockdown or overexpression of AGAP2-AS1 in vitro. Lastly, a biological mechanistic explanation was proposed as a result of our attempts to establish how AGAP2-AS1 regulates its prospective upstream/downstream transcription of genes.

| Ethics statement
Human pre-eclampsia placental tissues and normal placental samples were collected from the department of Obstetrics at Jiangsu Province Hospital, Jiangsu, China. The study was authorized by the hospital ethics board. Written informed consent was obtained from patients who met the inclusion criteria. The clinicopathological characteristics of each participant are summarized in Table 1.

| Transfections of cell lines
Suspend transfection for HTR-8/SVneo cells: 1-2 μL Lipofectamine 3000 were thoroughly mixed with 25 μL OPTI-MEM. In parallel, 20pmol of siRNAs were mixed with 25 μL OPTI-MEM through pipette. After incubation at room temperature for 5 minutes, the corresponding tubes (contained siRNAs and Lipo3000) were thoroughly mixed through moderate pipetting and then incubated 5-10 minutes at room temperature. At the end of incubation, the 50 μL transfection solution was used to re-suspend cell pellet (6 × 10 4 cells/each 24-well scale). After incubated 10 minutes at room temperature, 450 μL fresh complement medium was added to each tube and then the suspended cells were pipetted onto the culture plate.
On-plate transfections for primary trophoblast cells: 6 × 10 4 cells were plated on the 6-well plate scale. After on-plated 6 hours of primary cells, we performed the on-plated transfection assays. 100 pmol (10 μL) siRNAs were mixed with 240 μL OPTI-MEM by gentle pipetting and incubation for 5min. At the same time, 10 μL

| Recombinant plasmid construction
To ectopically up-regulate the expression of AGAP2-AS1, the AGAP2-AS1 plasmid (1567 bp, NR_027032.1, synthesized by Realgene, Nanjing, China) was subcloned into a pcDNA3.1(+) vector (Invitrogen), following standard protocols. AGAP2-AS1 plasmids as well as empty vectors were transfected into HTR-8/SVneo cells on 6-well plates and/or 24-well plates. SYBR Premix Ex Taq (TaKaRa) was used to examine the expression of AGAP2-AS1 and JDP2, in accordance with the manufacturer's instructions. All data were normalized to GAPDH; the primer sequences are listed in Table S1.

| High-throughput sequencing analysis
High-throughput sequencing assays were performed as previously reported by Xu et al 7 Using human trophoblast cells, we performed an RNA-seq experiment to establish the mRNA-seq profiles after transfection with effective siRNAs against the AGAP2-AS1 mRNAseq library, which was established for sequencing and exploiting standard Illumina protocols. Subsequently, we evaluated the pathways that were activated by AGAP2-AS1 by analysing our data against the Gene Ontology (GO) database.

| Cell proliferation assays
To assess the cell growth ability of HTR-8/SVneo and JEG3 cell lines, MTT and EdU assays, respectively, were conducted. These experiments were carried out as per the method reported by Xu et al. 9 For the MTT assay, HTR-8/SVneo and JEG3 cells were transfected with mimics (NC or miRNA-574-5p and/or siRNAs, containing 3000 cells/ well) and subsequently plated in 96-well plates with five duplicates.
Cell viability was measured every 24 hours, following standard protocols. The absorbance was detected at 490 nm using the ELx800 Universal Microplate Reader (BioTek). For the EdU assays, cells were plated into 24 wells with 5 × 10 4 cells/well. We then used the 5-ethynyl-2-deoxyuridine labelling/detection kit (RiboBio) to estimate cell viability, according to the manufacturer's instructions.

| Flow cytometric analysis of cell cycle and apoptosis
These assays were conducted as previously described by Xu et al 10 After transfecting cells with either siRNAs or the plasmid, we performed fluorescein isothiocyanate (FITC)-annexin V and propidium iodide (PI) staining using the FITC-Annexin V Apoptosis Detection kit (BD Biosciences), following the manufacturer's instructions. The cell cycle level was determined by propidium oxide staining using the Cycle Test Plus DNA Reagent kit (BD Biosciences), following the protocol. Data were analysed using the FACScan flow cytometer.

| Migration and invasion assays
This experiment was carried out as previously described in Xu et al 10 Briefly, HTR-8/SVneo and JEG3 cells were transfected with specific siRNAs and/or plasmid; they were then re-suspended in RPMI-1640

| Fluorescence in situ hybridization
This experiment was conducted as defined earlier by Xu et al 10 Firstly  Table S1.

| Immunoblotting analysis
These assays were performed to detect protein levels as previously described in Xu et al 9 Each sample (5-10 µl) was loaded onto a 10% SDS gel and then analysed using Western blotting. The samples were incubated with specific antibodies (GAPDH from FOXP1 and JDP2 purchased from Proteintech) at 1:1000 concentration. GAPDH was used as a loading control. The intensity of the autoradiogram protein bands was quantified using the Quantity One software (Bio-Rad).

| Luciferase reporter assays
The assays were performed as previously described by Xu et al 10,17 The complementary DNA fragment containing the wild-type and mutant JDP2 3′-UTR was linked into the pGL3-basic vector (Promega).

| Statistical analysis
All statistical analysis and graph formation were performed with GraphPad Prism version 7.0 (GraphPad Software) and Adobe Photoshop CC2015. The results were analysed using an unpaired two-tailed t test and are presented as the mean ± standard error of the mean (SEM). Significance was noted at P < .01 (**), P < .05 (*).

| AGAP2-AS1 is down-regulated in the PE placenta and is related to the gestational age and body weight of infants
Firstly, we performed real-time PCR to detect AGAP2-AS1 expression in 20 normal placenta tissues and PE tissues. As shown in Figure 1A, we found that the expression of AGAP2-AS1 was significantly decreased in the PE group, compared to that in the control group. Then, we analysed the potential correlation between the expression of AGAP2-AS1 and the clinical features observed in patients. The clinical phenotypes are listed in Table 1. Notably, low AGAP2-AS1 expression was significantly correlated with the gestational age (P < .05) and the body weight of the infants (P < .05). In addition, there were significant differences in the diastolic blood pressure (P < .01), systolic blood pressure (P < .01) and body weight of infants (P < .05), while there were no differences observed in the maternal age or maternal weight between either the PE or the control group.

| AGAP2-AS1 affects proliferation and apoptosis of trophoblasts
As lncRNAs could have critical functions in diverse biological processes, we suspected that the aberrant reduction of AGAP2-AS1 in PE may affect biological processes, such as trophoblast cell migration, apoptosis and growth. Further, we postulated that these effects could be factors in the occurrence and development of PE. Thus, we explored the potential function and mechanism of AGAP2-AS1 in trophoblasts.
We tested the basic expression of AGAP2-AS1 in trophoblast cell lines. As shown in Figure 1D,  Figure 2E). Conversely, it was demonstrated that elevated expression of AGAP2-AS1 could reduce G0/ G1 phase accumulation ( Figure 2F). Further, flow cytometric analysis was also performed to assess whether aberrant expression of AGAP2-AS1 can affect trophoblast cell apoptosis. As shown in Figure 2G, the ratio of the total apoptotic cells was significantly increased after transfection with AGAP2-AS1-specific siRNAs (siAGAP2-AS1) in vitro. Similarly, the protein level of cleaved caspase3 was evaluated; the data showed that knockdown of AGAP2-AS1 promoted apoptosis. Thus, these noticeable findings indicate that AGAP2-AS1 positively affects trophoblast cell growth and, contrastingly, negatively affects cell apoptosis.

| AGAP2-AS1 affects migration and invasion of trophoblasts
As previously noted, the abnormal ability of trophoblast migration and invasion cannot effectively induce spiral artery remodelling; moreover, its occurrence drives the progression of PE. Therefore, we performed Transwell assays to identify the effects on migration and invasion after AGAP2-AS1 knockdown or overexpression. The results showed that the silencing of AGAP2-AS1 in trophoblast cells can inhibit cell migration and invasion ( Figure 3A). Conversely, we showed that up-regulating the expression of AGAP2-AS1 can induce cell migration and invasion ( Figure 3B). Collectively, the data presented here highlight that AGAP2-AS1 overexpression positively affects trophoblast cell invasion and migration.

| AGAP2-AS1 can be activated by the transcription factor FOXP1
Previous studies have reported that the majority of human lncRNA expressions can be mediated by various transcription factors. 18,19 Thus, we first used the JASPAR database (http://jaspa r2014.gener eg.net) to explore the latent factor that activated or inactivated the expression of AGAP2-AS1. As shown in Table 2, 17 FOXP1-binding sites were predicted in the promoter region of AGAP2-AS1, which implied that AGAP2-AS1 might be regulated by the transcription factor FOXP1 in trophoblast cells. We then used specific siRNAs and plasmids either down-regulate or up-regulate FOXP1 at the protein and RNA levels in the HTR-8/SVneo cells, respectively ( Figure 4A,B).
The expression of AGAP2-AS1 was significantly decreased after the silencing of FOXP1, whereas the levels of AGAP2-AS1 were dramatically increased upon the overexpression of FOXP1 ( Figure 4C,D).
We then performed ChIP assays and the results indicated that transcription factor FOXP1 could bind to the promoter region to stimulate AGAP2-AS1 expression at the RNA level ( Figure 4E). The data presented here highlight that AGAP2-AS1 can be activated by the transcription factor FOXP1.

| Gene expression profiling
To clarify the molecular mechanism of AGAP2-AS1-mediated regulation, the silencing of AGAP2-AS1 was performed in human  Figure 5D). Subsequently, the results of Western blot analysis showed that the changes at the protein level were consistent with the mRNA results ( Figure 5E; Tables S2 and S3).

| AGAP2-AS1 regulates JDP2 expression through the AGAP2-AS1/miR-574 axis
To further explore the latent mechanisms of AGAP2-AS1-mediated regulation in trophoblasts, we first conducted the fluorescence in  and JDP2 expression at both the RNA ( Figure 6D,E) and protein levels ( Figure 6F,G). Furthermore, we constructed diverse luciferase genes, including AGAP2-AS1, mutant AGAP2-AS1, 3'-UTR of JDP2 and mutant 3'-UTR of JDP2, cotransfected with miR-574 mimics ( Figure 6H,I). Through the construction of these diverse luciferase genes, we demonstrated that the relative luciferase activities of AGAP2-AS1 and 3' UTR of JDP2 were eliminated when the cells were transfected with the miR-574 mimics. Conversely, there was no effect observed in either the mutant AGAP2-AS1 or the mutant 3′UTR of JDP2 group. Thus, the resulting data validated the fact that miR-574 can directly bind to both AGAP2-AS1 and JDP2. Further, we demonstrated that AGAP2-AS1 regulates JDP2 expression via the AGAP2-AS1/miR-574 axis in trophoblasts.

| D ISCUSS I ON
To date, there has been an abundance of evidence that highlights how the aberrant expression of lncRNAs could be involved in various human disorders, including PE. 22 Our previous studies have shown that the down-regulated expression of TUG1 7 or HOXA11-AS 10 could significantly inhibit trophoblasts migration and proliferation and promote cell apoptosis in vitro. Additionally, other lncRNAs, such as HOTAIR, 23 GHET1 24 and TCL6, 25 are reportedly able to regulate the trophoblastic phenotype that further accelerates the pathogenesis of PE. Thus, we need to identify more prospective PE-related lncRNAs and further explore their biological functions and mechanisms in the context of PE.
In various reports, the association of AGAP2-AS1 with numerous diseases, such as pancreatic cancer, 26 glioblastoma 27 and breast cancer 15 has been identified. In this work, we found that lncRNA AGAP2-AS1 is significantly down-regulated in pre-eclamptic placenta compared with that in the control group and that the lower expression of AGAP2-AS1 is negatively correlated with the birth weight and gestational age of infants, indicating that down-regulation of AGAP2-AS1 is positively related to early termination of pregnancy, which further suggests that down-regulation of AGAP2-AS1 might lead to severe PE. We conducted specific functional assays to explore the potential implications of these findings. Up-regulated AGAP2-AS1 expression could promote trophoblast proliferation, invasion, and migration and inhibit cell apoptosis in trophoblasts.
Conversely, the silencing of AGAP2-AS1 stimulated cell apoptosis and reduced cell growth and invasion, thus establishing that the knockdown of AGAP2-AS1 expression exerts inhibitory effects on trophoblasts by impairing the cellular phenotype, including cell growth, apoptosis and invasion. Thus, these results demonstrated that AGAP2-AS1 might act as a molecular marker for the screening of PE.
Next, we performed the RNA-seq (RNA transcriptome sequencing) assay to explore the potential downstream targets of AGAP2-AS1.
After the silencing of AGAP2-AS1, we discovered a noticeable decrease in both the mRNA and protein levels of the cellular activator JDP2.
JDP2, a member the activator protein family, has been implicated in F I G U R E 4 AGAP2-AS1 can be activated by transcript factor FOXP1. A and B, FOXP1 expression level at protein and RNA level by Western blotting and qPCR, respectively, when HTR-8/SVneo cells were transfected with FOXP1-specific siRNAs. The AGAP2-AS1 expression level was tested by qPCR after treated with siRNAs against AGAP2-AS1 (B, right panels). C and D, FOXP1 expression level at protein and RNA level by Western blotting and qPCR, respectively, when HTR-8/SVneo cells were transfected with FOXP1 plasmid. The AGAP2-AS1 expression level was tested by qPCR after treated with plasmid (D, right panels). E, ChIP-qPCR of FOXP1 bind to the promoter of AGAP2-AS1 in HTR-8/SVneo, IgG as negative control. **P < .01, *P < .05 many cellular processes including migration, invasion and proliferation. 28 F I G U R E 6 AGAP2-AS1 regulates JDP2 expression by AGAP2-AS1/miR-574 axis. A, AGAP2-AS1 location was detected by FISH in HTR-8/ SVneo. Red, AGAP2-AS1; blue, DAPI. B, Bioinformatics analysis exhibited the binding probability between JDP2 and miR-574. C, The RNAup algorithm predicted latent binding of miR-574 to AGAP2-AS1 and JDP2, with conservative sequence in the 3′-UTR regions. D and E, HTR-8/ SVneo cells were treated with siCon or si-AGAP2-AS1, mimics NC or mimics miR-574, inhibit NC or inhibitor miR-574. Using RT-PCR and Western blotting analysis to detect miR-574 (on the left), AGAP2-AS1 (in the middle) and JDP2 expression (on the right), respectively. F and G, Western blotting analysis was performed to test JDP2 expression at protein level after treated with mimics NC or mimics miR-574, inhibit NC or inhibitor miR-574. H, Luciferase reporter assays were conducted to detect the interacting activity between miR-574 with AGAP2-AS1/JDP2, normalized by Renilla activity. Numbers are shown as the mean ± SEM, **P < .01, *P < .05 in trophoblasts. Overall, these results established the fact that AGAP2-AS1 can be activated by FOXP1, further indicating that lncRNAs can be regulated simultaneously by many different transcriptional factors.
To conclude, in this report, we highlighted that the biological role and mechanism of AGAP2-

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this article.