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Dysfunction of pseudogene PGK1P2 is involved in preeclampsia by acting as a competing endogenous RNA of PGK1
Pregnancy Hypertension 13 (2018) 37–45
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Pregnancy Hypertension journal homepage: www.elsevier.com/locate/preghy
Dysfunction of pseudogene PGK1P2 is involved in preeclampsia by acting as a competing endogenous RNA of PGK1 ⁎
Jing Tonga,b, Jieqiong Yanga,b, Hong Lva,b, Shijian Lva,b, Cong Zhanga,b,c, , Zi-Jiang Chena,b,
T
⁎
a
Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200135 China Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai 200135 China c Shandong Provincial Key Laboratory of Animal Resistance Biology, College of Life Sciences, Shandong Normal University, 88 East Wenhua Road, Ji'nan, Shandong 250014 China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Preeclampsia Competing endogenous RNA PGK1 PGK1P2 LncRNA miRNA-330-5p
Objectives: Normal decidualization is essential for normal pregnancy and abnormal decidualization is thought to cause preeclampsia (PE). Phosphoglycerate kinase 1 (PGK1) is an enzyme involved in the glycolytic pathway which is the main metabolism process decidual cells exhibit. Phosphoglycerate kinase 1, pseudogene 2 (PGK1P2), which is also a long non-coding RNA (lncRNA), has a high sequence similarity to PGK1 and therefore acquires an ability for sequence-specific regulation. Methodology: The expression of PGK1 and PGK1P2 in human decidua, as well as their relationship and functions during decidualization was investigated using in vitro cultured human endometrial stromal cell lines (hESCs) and primary ESCs by real-time PCR, immunohistochemistry, western blotting, siRNA techniques and miRNA inhibitor or mimic transfection. Results: The levels of PGK1 and PGK1P2 mRNA and PGK1 protein in severe preeclamptic decidua were lower than those in normal pregnant controls. PGK1 and PGK1P2 mRNAs were both induced after in vitro decidualization and their deficiency caused impaired decidualization in turn. We also found PGK1P2 acted as a competing endogenous RNA (ceRNA) to regulate PGK1 expression through miR-330-5p. Conclusions: We proved that PGK1 and PGK1P2 are a pair of ceRNAs against miR-330-5p and they play a vital role in human decidualization by regulating angiogenesis and glycolysis metabolism. The deficiency of PGK1 and PGK1P2 in the decidua jeopardizes the decidualization process and subsequently might lead to the occurrence of PE. These findings may help in promoting novel predictive, diagnostic and prognostic biomarkers of PE in future.
1. Introduction Preeclampsia (PE) is a disease unique to human pregnancy characterized by hypertension and/or proteinuria [1]. It is the major contributor to maternal death and perinatal mortality and morbidity worldwide and causes socioeconomic burden to the affected families and society consequently. To date, delivery of the placenta is the only effective treatment for PE. Most of the symptoms will disappear within a week after delivery. However, the pathogenesis of PE remains an enigma. It is widely believed that the genesis of preeclampsia resides in the placental bed during early pregnancy. Impaired implantation and deficient placentation in early pregnancy leads to disturbed placental function which is thought to be the major cause of PE. Deficient
placentation is characteristic of PE, but the underlying causes are unknown [2]. In normal human placentation, uterine invasion by trophoblast cells and subsequent spiral artery remodeling depend on establishing a talk between fetal trophoblasts and maternal decidua. The major component of placental bed is the decidua into which trophoblasts invade [3]. Both successful embryo implantation and placentation relies on maternal adequate decidualization of endometrial stromal cells (ESCs) [4]. The extent of decidualization appears to correlate with the degree of trophoblast invasion and placenta formation. Accumulating attention has been given to decidua in PE researches. Impaired decidualization causes failed intravascular trophoblast invasion and deficient placentation which increases the risk of PE [5,6]. Furthermore, several
Abbreviations: PE, preeclampsia; ESCs, endometrial stromal cells; hESCs, human endometrial stromal cell lines; PGK1, phosphoglycerate kinase 1; PGK1P2, phosphoglycerate kinase 1, pseudogene 2; LncRNA, long non-coding RNA; ceRNA, competing endogenous RNA; SPE, severe preeclampsia; NP, normal pregnancies; VEGF, vascular endothelial growth factor; PRL, prolactin; IGFBP1, insulin-like growth factor binding protein 1 ⁎ Corresponding authors at: Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200135 China. E-mail addresses: [email protected] (C. Zhang), [email protected] (Z.-J. Chen). https://doi.org/10.1016/j.preghy.2018.05.003 Received 26 October 2017; Received in revised form 21 April 2018; Accepted 2 May 2018 Available online 03 May 2018 2210-7789/ © 2018 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.
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developed by the diaminobenzidine (DAB)-HRP reaction system.
knock-out mouse models have revealed that endometrial decidualization is vital for maintaining pregnancy [7,8]. Naturally normal decidualization is essential for normal pregnancy and abnormal decidualization caused by disorders of decidual tissues is thought to cause PE [6,9]. PGK1 (phosphoglycerate kinase 1) has traditionally been studied as an important adenosine triphosphate (ATP)-generating enzyme of the glycolytic pathway in which it catalyzes the conversion of 1, 3-diphosphoglycerate to 3-phosphoglycerate [10]. Furthermore, PGK1 can influence DNA replication and repair in the nucleus [11,12], it can be secreted extracellularly by tumors such as prostate cancer [13]. In cancer researches, PGK1 is found to be correlated with peritoneal dissemination in gastric cancer [10] and participates in the angiogenic process as a disulphide reductase [14]. PGK1P2 (phosphoglycerate kinase 1, pseudogene 2), one of PGK pseudogenes, is a long non-coding RNA (LncRNA), it has a high sequence similarity to its parental proteincoding gene PGK1. Our previous research has demonstrated both PGK1 and PGK1P2 significantly decreased in the decidua of PE patients [15], however, their transcription and function in maternal decidua remains largely unknown. Pseudogenes as well as LncRNAs are traditionally claimed to not yield functional mRNAs and not translated into proteins consequently, thus regarded as garbage fragments or dark matter in the genome, however, recent studies have demonstrated that they can regulate gene function in many ways [16]. Specifically, pseudogene and LncRNA transcripts can act as natural microRNA (miRNA) sponges to suppress intracellular miRNA function by using shared miRNA response elements, like competing endogenous RNA (ceRNA) does [17]. Its interaction with miR-330-5p has already been identified from a HITS-CLIP data [18]. Taken together, these previous observations encouraged us to investigate the role of PGK1 and PGK1P2 in decidualization process and their relationship with PE. We also focused on whether PGK1P2 can act as a ceRNA for PGK1 against miR-330-5p.
2.3. Cell culture and reagents The immortalized human endometrial stromal cell lines (hESCs) was a kind gift from Dr. Haibin Wang (Xiamen University Medical College, China). The hESCs were cultured in Phenol Red-free DMEM/F12 containing glutamine (Life Technologies, Inc., Grand Island, NY), 10% dextran-coated charcoal-stripped FBS (Biological Industries, Beit Haemek, Israel), 5 × 10−2 g/L antibiotics (Gibco, Grand Island, NY, USA), 5 × 10−4 g/L puromycin (Gibco, Grand Island, NY, USA) and 1% Insulin-Transferrin-Selenium (Invitrogen, Carlsbad, CA, USA) at 37 °C in a humidified atmosphere of 5% CO2 in air. 2.4. Isolation and culture of primary human endometrial stromal cells (ESCs) Endometrial tissues were obtained by dilatation and curettage between day 7–12 of the menstrual cycle from women with regular menstrual cycles and no apparent endometrial dysfunction at the time of diagnostic hysteroscopy. Primary ESCs were isolated from those proliferative endometrial tissues by enzymatic digestion and filtration which produces > 95% pure stromal cell cultures as described previously [21,22]. Briefly, tissues were washed twice with DMEM/F12 medium (Life Technologies, Inc., Grand Island, NY) and finely minced with scissors. Then they were digested by collagenase type I (c0130; Sigma Chemical Co., St. Louis, MO, USA) for 70 min at 37 °C and digested with deoxyribonuclease (DN25; Sigma Chemical Co., St. Louis, MO, USA) for 20 min at 37 °C. Then the digested tissues were filtered through 180 and 40 μm griddles sequentially to remove mucous and undigested tissues and to permit stromal cells to pass. Primary ESCs were seeded in 75 cm2 tissue culture flasks and cultured in the same condition as hESCs did but free of 1% Insulin- Transferrin- Selenium.
2. Methodology 2.5. In vitro decidualization and siRNA transfection
2.1. Study population and decidual sample collection
Cells were inoculated into 6-well plates (106 cells per well) for future manipulation. For in vitro decidualization inducing, the FBS’s concentration were reduced to 2% (v/v) and the cells were treated with 10−6 mol/L medroxyprogesterone-17-acetate (MPA) (Sigma Chemical Co., St. Louis, MO, USA) dissolved in ethanol, and 5 × 10−4 mol/L N6, 2′-O-dibutyryladenosine cAMP sodium salt (cAMP) (Sigma Chemical Co., St. Louis, MO, USA) for 6 days. While control samples were only treated with 0.1% (v/v) ethanol. As for siRNA transfection, cells were incubated in the antibiotics free medium, at 60% confluence, Lipofectamine 3000 Transfection Reagent (7.5 μl/well; Invitrogen) and 50 nM PGK1-siRNA (RiboBio, Guangzhou, China), PGK1P2-siRNA (GenePharma, Shanghai, China) or non-targeting control siRNA diluted in Opti-MEM (Invitrogen) were transfected to hESCs or primary ESCs. The medium was changed 24 h later, then the cells were induced to decidualization for 4 days.
Sixteen women with pregnancies complicated by severe preeclampsia (SPE) and sixteen women with normal pregnancies (NP) were recruited from the Department of Obstetrics and Gynecology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University between April 2015 and March 2016. SPE was defined as systolic blood pressure ≥160 mm Hg or diastolic blood pressure ≥110 mm Hg and/or proteinuria > 3+ protein on dip stick [19]. Only pregnancies delivered by caesarean section were included and none of them were in labor prior to caesarean section. Women with chronic hypertension, diabetes mellitus, renal disease, reduced thyroid, fetal and placental structural abnormalities, and other pregnancy related complications were excluded. Decidual tissues were scrubbed from the placental bed at the time of caesarean section. The tissues were washed with sterilized water to remove blood, snap frozen in liquid nitrogen immediately and then were stored at −80 °C until use. The study was approved by the Ren Ji Hospital Research and Ethics Committees. Informed consent was obtained from all participants before the collection of decidual tissues.
2.6. Transient transfection For the transfection of miR-330-5p mimic and inhibitor, hESCs and primary ESCs were seeded in 6-well plates (5 × 105 cells per well) with antibiotics free medium. Lipofectamine3000 Transfection was mixed with 50 nM miR-330-5p mimic, 100 nM miR-330-5p inhibitor or scrambled control (RiboBio, Guangzhou, China). The mixture was then added to hESCs or primary ESCs at 50% confluence. 6 h later, the medium was changed and the cells were induced to decidualization. As for hESCs, the in vitro decidualization lasted 4 days. While for primary ESCs, the in vitro decidualization continued 4 days for miR-330-5p mimic and 3 days for transfection of miR-330-5p inhibitor.
2.2. Immunohistochemistry PGK1 expression was studied by immunohistochemistry (IHC) on paraffin-embedded sections as described previously [20]. Tissue sections were deparaffinized and microwaved for 10 min in 10 mM sodium citrate buffer (PH 6.0) to retrieve antigen. Sections were blocked for endogenous peroxidase activity and incubated with anti-PGK1 (178111-AP, Proteintech) overnight at 4 °C. Subsequently, the sections were incubated with appropriate HRP-conjugated secondary antibody and 38
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Fig. 1. Expression of PGK1 and PGK1P2 in decidual tissues of NP and SPE subjects. A The mRNA expression of PGK1 and PGK1P2 in the decidual tissues from the NP and SPE groups. B The protein expression of PGK1 in the decidual tissues from the NP and SPE groups. C The relative expression levels of the PGK1 protein compared to that of β-actin (n = 16 for each group). D–G Location of PGK1 protein in decidual tissues of NP and SPE subjects. (d1), (d2), (f1) and (f2) depict the decidual sections from the NP group. (e1), (e2), (g1) and (g2) are from the SPE group. NP, normal pregnancy group. SPE, severe preeclampsia group. NC, negative control. The data are presented as the means ± SEM. Statistical significance is represented as follows: * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bars = 50 μm.
0.01% (w/v) bromophenol blue and 5% (w/v) β-mercaptoethanol was boiled for 5 min at 100 °C. The samples were run on 10% (v/v) SDSpolyacrylamide gel and transferred onto nitrocellulose (NC) blotting membranes. Then the membranes were blocked with 5% (w/v) nonfat milk powder at room temperature for 1.5 h followed by an incubation with primary antibodies against PGK1 (ab38007, Abcam; 1:100 dilution), or β-actin (SC-47778, Santa Cruz Biotechnology; 1:5000 dilution) at 4 °C overnight. After washed by Tris-buffered saline containing Tween 20 three times, the membranes were incubated with horseradish peroxidase-conjugated IgG antibody (1:5000 dilution) at room temperature for 1 h. At the end, the bands were detected after incubation with Immobilon™ Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA) using an Enhanced Chemiluminescence Western Blotting Detection System (Tanon Science & Technology Co., Ltd., Shanghai, China). Results from three separate experiments were analyzed by QuantiScan software (Biosoft, Cambridge, UK) and the relative densities of the detected bands were calculated by normalizing to the densities of β-actin.
2.7. RNA isolation and quantitative real-time PCR (QPCR) Total RNA was isolated by using an Animal Total RNA Isolation Kit (Foregene, Chengdu, China) referring to the manufacturer’s instructions. RNA sample (500 ng) was converted to cDNA using the PrimerScript TM RT Master Mix (Takara, Japan). As for miRNA, RNA sample (400 ng) was converted to cDNA using the Mir-X miRNA FirstStrand Synthesis Kit (Takara, Japan). QPCR was performed using SYBR Green master mix (Toyobo, Japan) in Vii A TM 7 Real-time PCR system (Life Technologies, Inc., Grand Island, NY) under the following conditions: 3 min at 95 °C; 40 cycles of 15 s at 95 °C, 30 s at 60 °C, 30 s at 72 °C; and 5 min at 72 °C. For a quantitative measurement, β-actin or U6 snRNA was used as an internal control. Relative mRNA and miRNA level was determined using the ΔΔCt method [23]. All QPCR reactions were performed in triplicate. The primer sequences are listed in Table S2. 2.8. Western blot analysis Total protein was isolated from decidual tissues, hESCs and primary ESCs using RIPA buffer (50 mM TRIS pH 7.4, 150 mM NaCl, 1% [v/v] Triton™ X-100, 1% [w/v] sodium deoxycholate, 0.1% [w/v] sodium dodecyl sulfate [SDS]) mixed with protease inhibitors (Sigma Chemical Co.). The isolated proteins were quantified with an Enhanced BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). A solution containing 20 μg protein solubilized in a sample buffer consisting of 62.5 mM Tris- HCl pH 6.8, 2% (w/v) SDS, 25% (v/v) glycerol,
2.9. Statistical analysis All statistical analyses were performed using SPSS statistical software (IBM, Chicago, Illinois, USA). The differences between two groups were analyzed by Student’s t test. All data are expressed as means ± SEM and p < 0.05 was considered significant. All experiments were repeated at least three independent times. 39
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Fig. 2. Expression of decidualization-related genes in hESCs and primary ESCs after induced decidualization for 3 and 6 days. A–C are the experimental results of hESCs, D–F are the experimental results of primary ESCs. A, D The mRNA expression of PGK1, PGK1P2, PRL and IGFBP1 after induction of decidualization. B, E Bands representing PGK1 and β-actin proteins on a western blot. C, F The relative expression levels of PGK1 compared to that of β-actin. The data are presented as the mean ± SEM. Statistical significance is represented as follows: * p < 0.05, ** p < 0.01, *** p < 0.001. Ctrl, control hESCs or control primary ESCs; cAMP + MPA, treated hESCs or treated primary ESCs. PRL, prolactin; IGFBP1, insulin-like growth factor binding protein 1.
3. Results
protein expressed in both NP and SPE decidual tissues compared to negative control (Fig. 1D–G). But the PGK1 protein was less abundant in SPE group compared to NP group.
3.1. Clinical characteristics of the study population The clinical information of all participants in the study was summarized in Table S1. The SPE and NP controls were matched for maternal age. Nonetheless, women in SPE group delivered newborns with lower gestational age at birth and birth weight (p < 0.01). As expected, the systolic blood pressure, diastolic blood pressure was higher in SPE group (p < 0.01).
3.3. PGK1 and PGK1P2 are both involved in decidualization induced in vitro In vitro decidualization of hESCs and primary ESCs was performed to investigate the involvement of PGK1 and PGK1P2 in this process. The variant cellular morphology resulting from the induction treatments is shown in Figs. S1 and S2. The hESCs and primary ESCs became rounded, relatively large epithelioid-like or polygonal decidual cells after treatment with cAMP and MPA compared to the fibroblast like phenotype in the control sample, and this transformation was time dependent. Moreover, the detection of notable increase of endometrial decidualization biomarkers, prolactin (PRL) and insulin-like growth factor binding protein1 (IGFBP1) indicated the cells were decidulized after the induction (Fig. 2A). During decidualization, the mRNA levels of PGK1 and PGK1P2 increased markedly compared to those of the controls (Fig. 2A and D). The PGK1 protein level showed the same
3.2. Expression of PGK1 and PGK1P2 in decidual tissues of NP and SPE Decidual tissues from 16 NP and 16 SPE were examined for the expression of PGK1 and PGK1P2 by qPCR and Western Blot. The mRNA levels of PGK1 and PGK1P2 in the SPE patients were significantly lower compared to those in NP (P < 0.001) (Fig. 1A). Also, a significant decrease in PGK1 protein level could be found in the SPE group compared to the NP group (Fig. 1B, and C). Immunohistochemistry was performed to locate the PGK1 protein. The results showed that PGK1 40
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Fig. 3. Effects of PGK1 depletion by siRNA on in vitro decidualization of hESCs and primary ESCs. A–C are the experimental results of hESCs, D–F are the experimental results of primary ESCs. A, D The mRNA expression of PGK1, PGK1P2, VEGF, PRL and IGFBP1 after the down regulation of PGK1. B, E Bands representing PGK1 and β-actin proteins. C, F The relative expression levels of PGK1 compared to that of β-actin. The data are presented as the means ± SEM. Statistical significance is represented as follows: * p < 0.05, ** p < 0.01, *** p < 0.001. PRL, prolactin; IGFBP1, insulin-like growth factor binding protein 1.
3.5. PGK1 and PGK1P2 are targeted by miR-330-5p
pattern of response to in vitro decidualization by western blot analysis shown in Fig. 2B, C, E and F.
An important corollary of the ceRNA hypothesis is that ceRNAs coordinately regulate each other’s expression [17]. Since PGK1 and PGK1P2 increased simultaneously during in vitro decidualization (Fig. 2A and D), and PGK1P2 knockdown resulted in decreased PGK1 mRNA and protein abundance (Fig. 4), mirroring the PGK1P2 mRNA reduction obtained by PGK1-siRNA transfection (Fig. 3). This coexpression indicated that PGK1P2 might act as a ceRNA for PGK1 against some miRNAs. Previous study has already indicated PGK1P2 is a target of miR-330-5p [18]. “TargetScan Human” also showed that miR-330-5p seed matches within the high homology region that are conserved between PGK1 and PGK1P2 (Fig. S3A). So, we transfected PGK1P2-siRNA to hESCs and primary ESCs, the qPCR analyses revealed that the miR330-5p levels was increased by the knockdown of PGK1P2 (Fig. S3B).
3.4. Knocking down of PGK1 and PGK1P2 affected decidualization To further elucidate the function of PGK1 and PGK1P2 during the in vitro decidualization of hESCs and primary ESCs, we used RNA interference technology to down regulate the mRNA level of PGK1 and PGK1P2. The siRNA targeting PGK1 mRNA, PGK1P2 mRNA or a nontargeting siRNA were transfected into hESCs or primary ESCs for 24 h and then subjected to differentiation. The interference rate of siRNA transfection achieved almost 90% as demonstrated in Figs. 3A, D and 4A, 4D. This knock down of PGK1 (Fig. 3) and PGK1P2 (Fig. 4) mRNA in hESCs and primary ESCs resulted in a significant reduction of the mRNAs corresponding to PRL and IGFBP1 at the same time. Meanwhile, the mRNA level of vascular endothelial growth factor (VEGF) was reduced in hESCs and primary ESCs treated with either PGK1-siRNA or PGK1P2-siRNA (Figs. 3A, 3D and 4A, D).
3.6. The mechanism of miR-330-5p mediated function To extend our study beyond the function of PGK1 and PGK1P2 on decidualization, miR-330-5p were further studied. MiR-330-5p mimic was transfected into the hESCs and primary ESCs, the cells were then 41
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Fig. 4. Effects of PGK1P2 depletion by siRNA on in vitro decidualization of hESCs and primary ESCs. A–C are the experimental results of hESCs. D–F are the experimental results of primary ESCs. A, D The mRNA expression of PGK1, PGK1P2, VEGF, PRL and IGFBP1 after the down regulation of PGK1P2. B, E Bands representing PGK1 and β-actin proteins. C, F The relative expression levels of PGK1 compared to that of β-actin. The data are presented as the means ± SEM. Statistical significance is represented as follows: * p < 0.05, ** p < 0.01, *** p < 0.001. PRL, prolactin; IGFBP1, insulin-like growth factor binding protein 1.
downregulated in human decidual tissues of SPE compared to those of NP group. In the in vitro culture system, the mRNA expressions of PGK1, PGK1P2 and the protein level of PGK1 increased significantly during decidualization and this augment was time dependent. When PGK1 and PGK1P2 were knocked down, the levels of PRL and IGFBP1 decreased sharply. More importantly, we found that PGK1P2 acted as a ceRNA to regulate PGK1 expression by acting as a sponge for miR-330-5p during human decidualization process. These findings demonstrate PGK1 and PGK1P2, a pair of ceRNAs linked by miR-330-5p, participate in human decidualization, and their deficiency in human decidua is associated with SPE. Lots of evidences have showed that regulatory modalities jointly exerted by LncRNAs and pseudogenes play vital role in human diseases. LncRNA is remarkable for its functional heterogeneity and can bind miRNA or a miRNA’s target sequence. Pseudogenes are copies of protein-coding genes and share a high sequence similarity with their parental genes which generate the potential for sequence-specific regulation [16]. Hence, a ceRNA hypothesis has been proposed. In this hypothesis, RNAs can crosstalk with each other by competing for shared miRNAs [17]. miRNAs, being endogenous small RNAs of 19–25 nucleotides, can regulate gene expression by destabilizing/ degrading mRNAs through imperfect base pairing to the “seed match” region in
induced to decidualization for 4 days. QPCR analysis revealed that miR330-5p suppressed the mRNA abundance of both PGK1P2 and PGK1, the mRNA level of PRL and IGFBP1 decreased significantly at the same time (Fig. 5A and D). In addition, the PGK1 protein level showed the same patterns by western blot analysis shown in Fig. 5B, C, E and F. To further test whether the expression level of PGK1P2 and PGK1 were linked by miR-330-5p, miR-330-5p inhibitor was transfected into hESCs and primary ESCs, the cells were then induced to decidualization for 4 days and 3 days respectively. QPCR analyses indicated that the transfection of miR-330-5p inhibitor not only increased PGK1P2 level but also increased PGK1 level in both hESCs and primary ESCs. Meanwhile the PRL and IGFBP1 mRNA level increased significantly (Fig. 6A and D). Moreover, the PGK1 protein abundance also increased as shown in Fig. 6B, C, E and F.
4. Discussion In this study, we focused on the roles of PGK1 and PGK1P2 in the decidualization of hESCs and primary ESCs and their relationship with SPE. We also explored the interrelation between PGK1 and PGK1P2 to extend our study. We demonstrated that PGK1 decreased significantly both at the RNA and protein levels, and PGK1P2 was significantly 42
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Fig. 5. Effects of miR-330-5p mimic transfection on in vitro decidualization of hESCs and primary ESCs. A–C are the experimental results of hESCs, D–F are the experimental results of primary ESCs. A, D The mRNA expression of PGK1, PGK1P2, PRL, IGFBP1 and miR-330-5p after miR-330-5p mimic transfection combined induced decidulization. B, E Bands representing PGK1 and β-actin proteins. C, F The relative expression levels of PGK1 compared to that of β-actin. The data are presented as the means ± SEM. Statistical significance is represented as follows: * p < 0.05, ** p < 0.01, *** p < 0.001. PRL, prolactin; IGFBP1, insulin-like growth factor binding protein 1.
remodeling consequently [3]. Disturbances of angiogenesis signal pathways have been proved to be associated with PE [33]. VEGF is famous as an angiogenesis factor in many tissues and its decreased expression has been implicated in the pathophysiology of PE [34,35]. Researches have demonstrated extracellular PGK1 in prostate cancer causes a reduction of plasmin which can facilitate the cleavage of plasminogen to generate the vascular inhibitor angiostatin, which is known as an important regulator of an “angiogenic switch” [13]. In our study, we revealed the expression of VEGF decreased markedly with the down-regulated expression of PGK1, also providing a potential risk for decidual impairment. But further investigation exploring the relationship between PGK1 and VEGF is still needed. Furthermore, previous researches have provided unequivocal proof that glycolysis is activated in decidua for supporting early pregnancy when decidualization starts [20,36]. Decidual cells exhibit a high rate of glycolysis because it is an important source of precursors for synthesis of amino acids and lipids [37,38] and can provide sufficient energy for cell proliferation fast. The glycolysis-related genes such as GLUT1 [39] and PDK4 [40] are found to be induced during human decidualization and the impairment of glycolysis in placenta is found to be related with PE pathogenesis [38]. Therefore, we speculated as a key enzyme in the glycolytic pathway the deficiency of PGK1 in decidua might jeopardize glycolysis and subsequently contribute the
the 5’ UTR, CDS, or 3’ UTR of the mRNA [24]. Accumulating lines of evidence have led to the view that miR-330-5p is involved in the progression of prostate cancer [25], colorectal cancer [26] and cutaneous malignant melanoma [27]. In the present study, we have demonstrated that PGK1P2 was a ceRNA for PGK1 against miR-330-5p. This ceRNAbased regulatory circuitry plays important roles in post-transcriptional regulation [28] and represents a new phenomenon in PE physiological and pathological processes. In humans, decidualization is a response to progesterone during the secretory phase of menstrual cycle independent of the presence of an implanting blastocyst. At that time, ESCs transform and differentiate into decidualized stromal cells; decidual markers, such as PRL and IGFBP1, accumulate markedly [4]. As a consequence, decidualized stomal cells acquire the special ability to regulate trophoblast invasion and to resist inflammatory and oxidative abuse [29]. Obviously, decidualization is the preparation of the “maternal soil” for the “embryo seed” and is vital for embryo implantation and pregnancy maintenance [30]. The decidualization reaction is initiated around blood vessels [31]. It can promote maternal spiral arteries remodelling into high-capacitance vessels accompanied by enhanced expression of key angiogenic factors and microvascular angiogenesis [32]. The abnormalities of decidualization could compromise trophoblast invasion and spiral artery 43
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Fig. 6. Effects of miR-330-5p inhibitor transfection on in vitro decidualization of hESCs and primary ESCs. A–C are the experimental results of hESCs, D–F are the experimental results of primary ESCs. A, D The mRNA expression of PGK1, PGK1P2, PRL, IGFBP1 and miR-330-5p after miR-330-5p inhibitor transfection combined induced decidulization. B, E Bands representing PGK1 and β-actin proteins. C, F The relative expression levels of PGK1 compared to that of β-actin. The data are presented as the means ± SEM. Statistical significance is represented as follows: * p < 0.05, ** p < 0.01, *** p < 0.001. PRL, prolactin; IGFBP1, insulin-like growth factor binding protein 1.
Appendix A. Supplementary data
pathogenesis of PE. In summary, we speculate that PGK1 and PGK1P2 are a pair of ceRNAs against miR-330-5p and they play a vital role in human decidualization by regulating glycolysis metabolism and angiogenesis. The deficiency of PGK1 and PGK1P2 in the decidua jeopardizes the decidualization process, which subsequently leads to the occurrence of PE.
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Acknowledgements The authors would like to thank all the patients and their relatives for their participation during this study. This work was supported by the National Natural Science Foundation of China (NSFC: 31471399 and 31671199) and National Key R&D Program of China (2017YFC1001403) to C. Z.; by a grant from the Major Program of the National Natural Science Foundation of China (81490743) to Z.-J. C. and by the Shanghai Commission of Science and Technology (17DZ2271100).
Conflicts of interest There are no conflicts of interest. 44
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Contents lists available at ScienceDirect
Pregnancy Hypertension journal homepage: www.elsevier.com/locate/preghy
Dysfunction of pseudogene PGK1P2 is involved in preeclampsia by acting as a competing endogenous RNA of PGK1 ⁎
Jing Tonga,b, Jieqiong Yanga,b, Hong Lva,b, Shijian Lva,b, Cong Zhanga,b,c, , Zi-Jiang Chena,b,
T
⁎
a
Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200135 China Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai 200135 China c Shandong Provincial Key Laboratory of Animal Resistance Biology, College of Life Sciences, Shandong Normal University, 88 East Wenhua Road, Ji'nan, Shandong 250014 China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Preeclampsia Competing endogenous RNA PGK1 PGK1P2 LncRNA miRNA-330-5p
Objectives: Normal decidualization is essential for normal pregnancy and abnormal decidualization is thought to cause preeclampsia (PE). Phosphoglycerate kinase 1 (PGK1) is an enzyme involved in the glycolytic pathway which is the main metabolism process decidual cells exhibit. Phosphoglycerate kinase 1, pseudogene 2 (PGK1P2), which is also a long non-coding RNA (lncRNA), has a high sequence similarity to PGK1 and therefore acquires an ability for sequence-specific regulation. Methodology: The expression of PGK1 and PGK1P2 in human decidua, as well as their relationship and functions during decidualization was investigated using in vitro cultured human endometrial stromal cell lines (hESCs) and primary ESCs by real-time PCR, immunohistochemistry, western blotting, siRNA techniques and miRNA inhibitor or mimic transfection. Results: The levels of PGK1 and PGK1P2 mRNA and PGK1 protein in severe preeclamptic decidua were lower than those in normal pregnant controls. PGK1 and PGK1P2 mRNAs were both induced after in vitro decidualization and their deficiency caused impaired decidualization in turn. We also found PGK1P2 acted as a competing endogenous RNA (ceRNA) to regulate PGK1 expression through miR-330-5p. Conclusions: We proved that PGK1 and PGK1P2 are a pair of ceRNAs against miR-330-5p and they play a vital role in human decidualization by regulating angiogenesis and glycolysis metabolism. The deficiency of PGK1 and PGK1P2 in the decidua jeopardizes the decidualization process and subsequently might lead to the occurrence of PE. These findings may help in promoting novel predictive, diagnostic and prognostic biomarkers of PE in future.
1. Introduction Preeclampsia (PE) is a disease unique to human pregnancy characterized by hypertension and/or proteinuria [1]. It is the major contributor to maternal death and perinatal mortality and morbidity worldwide and causes socioeconomic burden to the affected families and society consequently. To date, delivery of the placenta is the only effective treatment for PE. Most of the symptoms will disappear within a week after delivery. However, the pathogenesis of PE remains an enigma. It is widely believed that the genesis of preeclampsia resides in the placental bed during early pregnancy. Impaired implantation and deficient placentation in early pregnancy leads to disturbed placental function which is thought to be the major cause of PE. Deficient
placentation is characteristic of PE, but the underlying causes are unknown [2]. In normal human placentation, uterine invasion by trophoblast cells and subsequent spiral artery remodeling depend on establishing a talk between fetal trophoblasts and maternal decidua. The major component of placental bed is the decidua into which trophoblasts invade [3]. Both successful embryo implantation and placentation relies on maternal adequate decidualization of endometrial stromal cells (ESCs) [4]. The extent of decidualization appears to correlate with the degree of trophoblast invasion and placenta formation. Accumulating attention has been given to decidua in PE researches. Impaired decidualization causes failed intravascular trophoblast invasion and deficient placentation which increases the risk of PE [5,6]. Furthermore, several
Abbreviations: PE, preeclampsia; ESCs, endometrial stromal cells; hESCs, human endometrial stromal cell lines; PGK1, phosphoglycerate kinase 1; PGK1P2, phosphoglycerate kinase 1, pseudogene 2; LncRNA, long non-coding RNA; ceRNA, competing endogenous RNA; SPE, severe preeclampsia; NP, normal pregnancies; VEGF, vascular endothelial growth factor; PRL, prolactin; IGFBP1, insulin-like growth factor binding protein 1 ⁎ Corresponding authors at: Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200135 China. E-mail addresses: [email protected] (C. Zhang), [email protected] (Z.-J. Chen). https://doi.org/10.1016/j.preghy.2018.05.003 Received 26 October 2017; Received in revised form 21 April 2018; Accepted 2 May 2018 Available online 03 May 2018 2210-7789/ © 2018 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.
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developed by the diaminobenzidine (DAB)-HRP reaction system.
knock-out mouse models have revealed that endometrial decidualization is vital for maintaining pregnancy [7,8]. Naturally normal decidualization is essential for normal pregnancy and abnormal decidualization caused by disorders of decidual tissues is thought to cause PE [6,9]. PGK1 (phosphoglycerate kinase 1) has traditionally been studied as an important adenosine triphosphate (ATP)-generating enzyme of the glycolytic pathway in which it catalyzes the conversion of 1, 3-diphosphoglycerate to 3-phosphoglycerate [10]. Furthermore, PGK1 can influence DNA replication and repair in the nucleus [11,12], it can be secreted extracellularly by tumors such as prostate cancer [13]. In cancer researches, PGK1 is found to be correlated with peritoneal dissemination in gastric cancer [10] and participates in the angiogenic process as a disulphide reductase [14]. PGK1P2 (phosphoglycerate kinase 1, pseudogene 2), one of PGK pseudogenes, is a long non-coding RNA (LncRNA), it has a high sequence similarity to its parental proteincoding gene PGK1. Our previous research has demonstrated both PGK1 and PGK1P2 significantly decreased in the decidua of PE patients [15], however, their transcription and function in maternal decidua remains largely unknown. Pseudogenes as well as LncRNAs are traditionally claimed to not yield functional mRNAs and not translated into proteins consequently, thus regarded as garbage fragments or dark matter in the genome, however, recent studies have demonstrated that they can regulate gene function in many ways [16]. Specifically, pseudogene and LncRNA transcripts can act as natural microRNA (miRNA) sponges to suppress intracellular miRNA function by using shared miRNA response elements, like competing endogenous RNA (ceRNA) does [17]. Its interaction with miR-330-5p has already been identified from a HITS-CLIP data [18]. Taken together, these previous observations encouraged us to investigate the role of PGK1 and PGK1P2 in decidualization process and their relationship with PE. We also focused on whether PGK1P2 can act as a ceRNA for PGK1 against miR-330-5p.
2.3. Cell culture and reagents The immortalized human endometrial stromal cell lines (hESCs) was a kind gift from Dr. Haibin Wang (Xiamen University Medical College, China). The hESCs were cultured in Phenol Red-free DMEM/F12 containing glutamine (Life Technologies, Inc., Grand Island, NY), 10% dextran-coated charcoal-stripped FBS (Biological Industries, Beit Haemek, Israel), 5 × 10−2 g/L antibiotics (Gibco, Grand Island, NY, USA), 5 × 10−4 g/L puromycin (Gibco, Grand Island, NY, USA) and 1% Insulin-Transferrin-Selenium (Invitrogen, Carlsbad, CA, USA) at 37 °C in a humidified atmosphere of 5% CO2 in air. 2.4. Isolation and culture of primary human endometrial stromal cells (ESCs) Endometrial tissues were obtained by dilatation and curettage between day 7–12 of the menstrual cycle from women with regular menstrual cycles and no apparent endometrial dysfunction at the time of diagnostic hysteroscopy. Primary ESCs were isolated from those proliferative endometrial tissues by enzymatic digestion and filtration which produces > 95% pure stromal cell cultures as described previously [21,22]. Briefly, tissues were washed twice with DMEM/F12 medium (Life Technologies, Inc., Grand Island, NY) and finely minced with scissors. Then they were digested by collagenase type I (c0130; Sigma Chemical Co., St. Louis, MO, USA) for 70 min at 37 °C and digested with deoxyribonuclease (DN25; Sigma Chemical Co., St. Louis, MO, USA) for 20 min at 37 °C. Then the digested tissues were filtered through 180 and 40 μm griddles sequentially to remove mucous and undigested tissues and to permit stromal cells to pass. Primary ESCs were seeded in 75 cm2 tissue culture flasks and cultured in the same condition as hESCs did but free of 1% Insulin- Transferrin- Selenium.
2. Methodology 2.5. In vitro decidualization and siRNA transfection
2.1. Study population and decidual sample collection
Cells were inoculated into 6-well plates (106 cells per well) for future manipulation. For in vitro decidualization inducing, the FBS’s concentration were reduced to 2% (v/v) and the cells were treated with 10−6 mol/L medroxyprogesterone-17-acetate (MPA) (Sigma Chemical Co., St. Louis, MO, USA) dissolved in ethanol, and 5 × 10−4 mol/L N6, 2′-O-dibutyryladenosine cAMP sodium salt (cAMP) (Sigma Chemical Co., St. Louis, MO, USA) for 6 days. While control samples were only treated with 0.1% (v/v) ethanol. As for siRNA transfection, cells were incubated in the antibiotics free medium, at 60% confluence, Lipofectamine 3000 Transfection Reagent (7.5 μl/well; Invitrogen) and 50 nM PGK1-siRNA (RiboBio, Guangzhou, China), PGK1P2-siRNA (GenePharma, Shanghai, China) or non-targeting control siRNA diluted in Opti-MEM (Invitrogen) were transfected to hESCs or primary ESCs. The medium was changed 24 h later, then the cells were induced to decidualization for 4 days.
Sixteen women with pregnancies complicated by severe preeclampsia (SPE) and sixteen women with normal pregnancies (NP) were recruited from the Department of Obstetrics and Gynecology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University between April 2015 and March 2016. SPE was defined as systolic blood pressure ≥160 mm Hg or diastolic blood pressure ≥110 mm Hg and/or proteinuria > 3+ protein on dip stick [19]. Only pregnancies delivered by caesarean section were included and none of them were in labor prior to caesarean section. Women with chronic hypertension, diabetes mellitus, renal disease, reduced thyroid, fetal and placental structural abnormalities, and other pregnancy related complications were excluded. Decidual tissues were scrubbed from the placental bed at the time of caesarean section. The tissues were washed with sterilized water to remove blood, snap frozen in liquid nitrogen immediately and then were stored at −80 °C until use. The study was approved by the Ren Ji Hospital Research and Ethics Committees. Informed consent was obtained from all participants before the collection of decidual tissues.
2.6. Transient transfection For the transfection of miR-330-5p mimic and inhibitor, hESCs and primary ESCs were seeded in 6-well plates (5 × 105 cells per well) with antibiotics free medium. Lipofectamine3000 Transfection was mixed with 50 nM miR-330-5p mimic, 100 nM miR-330-5p inhibitor or scrambled control (RiboBio, Guangzhou, China). The mixture was then added to hESCs or primary ESCs at 50% confluence. 6 h later, the medium was changed and the cells were induced to decidualization. As for hESCs, the in vitro decidualization lasted 4 days. While for primary ESCs, the in vitro decidualization continued 4 days for miR-330-5p mimic and 3 days for transfection of miR-330-5p inhibitor.
2.2. Immunohistochemistry PGK1 expression was studied by immunohistochemistry (IHC) on paraffin-embedded sections as described previously [20]. Tissue sections were deparaffinized and microwaved for 10 min in 10 mM sodium citrate buffer (PH 6.0) to retrieve antigen. Sections were blocked for endogenous peroxidase activity and incubated with anti-PGK1 (178111-AP, Proteintech) overnight at 4 °C. Subsequently, the sections were incubated with appropriate HRP-conjugated secondary antibody and 38
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Fig. 1. Expression of PGK1 and PGK1P2 in decidual tissues of NP and SPE subjects. A The mRNA expression of PGK1 and PGK1P2 in the decidual tissues from the NP and SPE groups. B The protein expression of PGK1 in the decidual tissues from the NP and SPE groups. C The relative expression levels of the PGK1 protein compared to that of β-actin (n = 16 for each group). D–G Location of PGK1 protein in decidual tissues of NP and SPE subjects. (d1), (d2), (f1) and (f2) depict the decidual sections from the NP group. (e1), (e2), (g1) and (g2) are from the SPE group. NP, normal pregnancy group. SPE, severe preeclampsia group. NC, negative control. The data are presented as the means ± SEM. Statistical significance is represented as follows: * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bars = 50 μm.
0.01% (w/v) bromophenol blue and 5% (w/v) β-mercaptoethanol was boiled for 5 min at 100 °C. The samples were run on 10% (v/v) SDSpolyacrylamide gel and transferred onto nitrocellulose (NC) blotting membranes. Then the membranes were blocked with 5% (w/v) nonfat milk powder at room temperature for 1.5 h followed by an incubation with primary antibodies against PGK1 (ab38007, Abcam; 1:100 dilution), or β-actin (SC-47778, Santa Cruz Biotechnology; 1:5000 dilution) at 4 °C overnight. After washed by Tris-buffered saline containing Tween 20 three times, the membranes were incubated with horseradish peroxidase-conjugated IgG antibody (1:5000 dilution) at room temperature for 1 h. At the end, the bands were detected after incubation with Immobilon™ Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA) using an Enhanced Chemiluminescence Western Blotting Detection System (Tanon Science & Technology Co., Ltd., Shanghai, China). Results from three separate experiments were analyzed by QuantiScan software (Biosoft, Cambridge, UK) and the relative densities of the detected bands were calculated by normalizing to the densities of β-actin.
2.7. RNA isolation and quantitative real-time PCR (QPCR) Total RNA was isolated by using an Animal Total RNA Isolation Kit (Foregene, Chengdu, China) referring to the manufacturer’s instructions. RNA sample (500 ng) was converted to cDNA using the PrimerScript TM RT Master Mix (Takara, Japan). As for miRNA, RNA sample (400 ng) was converted to cDNA using the Mir-X miRNA FirstStrand Synthesis Kit (Takara, Japan). QPCR was performed using SYBR Green master mix (Toyobo, Japan) in Vii A TM 7 Real-time PCR system (Life Technologies, Inc., Grand Island, NY) under the following conditions: 3 min at 95 °C; 40 cycles of 15 s at 95 °C, 30 s at 60 °C, 30 s at 72 °C; and 5 min at 72 °C. For a quantitative measurement, β-actin or U6 snRNA was used as an internal control. Relative mRNA and miRNA level was determined using the ΔΔCt method [23]. All QPCR reactions were performed in triplicate. The primer sequences are listed in Table S2. 2.8. Western blot analysis Total protein was isolated from decidual tissues, hESCs and primary ESCs using RIPA buffer (50 mM TRIS pH 7.4, 150 mM NaCl, 1% [v/v] Triton™ X-100, 1% [w/v] sodium deoxycholate, 0.1% [w/v] sodium dodecyl sulfate [SDS]) mixed with protease inhibitors (Sigma Chemical Co.). The isolated proteins were quantified with an Enhanced BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). A solution containing 20 μg protein solubilized in a sample buffer consisting of 62.5 mM Tris- HCl pH 6.8, 2% (w/v) SDS, 25% (v/v) glycerol,
2.9. Statistical analysis All statistical analyses were performed using SPSS statistical software (IBM, Chicago, Illinois, USA). The differences between two groups were analyzed by Student’s t test. All data are expressed as means ± SEM and p < 0.05 was considered significant. All experiments were repeated at least three independent times. 39
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Fig. 2. Expression of decidualization-related genes in hESCs and primary ESCs after induced decidualization for 3 and 6 days. A–C are the experimental results of hESCs, D–F are the experimental results of primary ESCs. A, D The mRNA expression of PGK1, PGK1P2, PRL and IGFBP1 after induction of decidualization. B, E Bands representing PGK1 and β-actin proteins on a western blot. C, F The relative expression levels of PGK1 compared to that of β-actin. The data are presented as the mean ± SEM. Statistical significance is represented as follows: * p < 0.05, ** p < 0.01, *** p < 0.001. Ctrl, control hESCs or control primary ESCs; cAMP + MPA, treated hESCs or treated primary ESCs. PRL, prolactin; IGFBP1, insulin-like growth factor binding protein 1.
3. Results
protein expressed in both NP and SPE decidual tissues compared to negative control (Fig. 1D–G). But the PGK1 protein was less abundant in SPE group compared to NP group.
3.1. Clinical characteristics of the study population The clinical information of all participants in the study was summarized in Table S1. The SPE and NP controls were matched for maternal age. Nonetheless, women in SPE group delivered newborns with lower gestational age at birth and birth weight (p < 0.01). As expected, the systolic blood pressure, diastolic blood pressure was higher in SPE group (p < 0.01).
3.3. PGK1 and PGK1P2 are both involved in decidualization induced in vitro In vitro decidualization of hESCs and primary ESCs was performed to investigate the involvement of PGK1 and PGK1P2 in this process. The variant cellular morphology resulting from the induction treatments is shown in Figs. S1 and S2. The hESCs and primary ESCs became rounded, relatively large epithelioid-like or polygonal decidual cells after treatment with cAMP and MPA compared to the fibroblast like phenotype in the control sample, and this transformation was time dependent. Moreover, the detection of notable increase of endometrial decidualization biomarkers, prolactin (PRL) and insulin-like growth factor binding protein1 (IGFBP1) indicated the cells were decidulized after the induction (Fig. 2A). During decidualization, the mRNA levels of PGK1 and PGK1P2 increased markedly compared to those of the controls (Fig. 2A and D). The PGK1 protein level showed the same
3.2. Expression of PGK1 and PGK1P2 in decidual tissues of NP and SPE Decidual tissues from 16 NP and 16 SPE were examined for the expression of PGK1 and PGK1P2 by qPCR and Western Blot. The mRNA levels of PGK1 and PGK1P2 in the SPE patients were significantly lower compared to those in NP (P < 0.001) (Fig. 1A). Also, a significant decrease in PGK1 protein level could be found in the SPE group compared to the NP group (Fig. 1B, and C). Immunohistochemistry was performed to locate the PGK1 protein. The results showed that PGK1 40
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Fig. 3. Effects of PGK1 depletion by siRNA on in vitro decidualization of hESCs and primary ESCs. A–C are the experimental results of hESCs, D–F are the experimental results of primary ESCs. A, D The mRNA expression of PGK1, PGK1P2, VEGF, PRL and IGFBP1 after the down regulation of PGK1. B, E Bands representing PGK1 and β-actin proteins. C, F The relative expression levels of PGK1 compared to that of β-actin. The data are presented as the means ± SEM. Statistical significance is represented as follows: * p < 0.05, ** p < 0.01, *** p < 0.001. PRL, prolactin; IGFBP1, insulin-like growth factor binding protein 1.
3.5. PGK1 and PGK1P2 are targeted by miR-330-5p
pattern of response to in vitro decidualization by western blot analysis shown in Fig. 2B, C, E and F.
An important corollary of the ceRNA hypothesis is that ceRNAs coordinately regulate each other’s expression [17]. Since PGK1 and PGK1P2 increased simultaneously during in vitro decidualization (Fig. 2A and D), and PGK1P2 knockdown resulted in decreased PGK1 mRNA and protein abundance (Fig. 4), mirroring the PGK1P2 mRNA reduction obtained by PGK1-siRNA transfection (Fig. 3). This coexpression indicated that PGK1P2 might act as a ceRNA for PGK1 against some miRNAs. Previous study has already indicated PGK1P2 is a target of miR-330-5p [18]. “TargetScan Human” also showed that miR-330-5p seed matches within the high homology region that are conserved between PGK1 and PGK1P2 (Fig. S3A). So, we transfected PGK1P2-siRNA to hESCs and primary ESCs, the qPCR analyses revealed that the miR330-5p levels was increased by the knockdown of PGK1P2 (Fig. S3B).
3.4. Knocking down of PGK1 and PGK1P2 affected decidualization To further elucidate the function of PGK1 and PGK1P2 during the in vitro decidualization of hESCs and primary ESCs, we used RNA interference technology to down regulate the mRNA level of PGK1 and PGK1P2. The siRNA targeting PGK1 mRNA, PGK1P2 mRNA or a nontargeting siRNA were transfected into hESCs or primary ESCs for 24 h and then subjected to differentiation. The interference rate of siRNA transfection achieved almost 90% as demonstrated in Figs. 3A, D and 4A, 4D. This knock down of PGK1 (Fig. 3) and PGK1P2 (Fig. 4) mRNA in hESCs and primary ESCs resulted in a significant reduction of the mRNAs corresponding to PRL and IGFBP1 at the same time. Meanwhile, the mRNA level of vascular endothelial growth factor (VEGF) was reduced in hESCs and primary ESCs treated with either PGK1-siRNA or PGK1P2-siRNA (Figs. 3A, 3D and 4A, D).
3.6. The mechanism of miR-330-5p mediated function To extend our study beyond the function of PGK1 and PGK1P2 on decidualization, miR-330-5p were further studied. MiR-330-5p mimic was transfected into the hESCs and primary ESCs, the cells were then 41
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Fig. 4. Effects of PGK1P2 depletion by siRNA on in vitro decidualization of hESCs and primary ESCs. A–C are the experimental results of hESCs. D–F are the experimental results of primary ESCs. A, D The mRNA expression of PGK1, PGK1P2, VEGF, PRL and IGFBP1 after the down regulation of PGK1P2. B, E Bands representing PGK1 and β-actin proteins. C, F The relative expression levels of PGK1 compared to that of β-actin. The data are presented as the means ± SEM. Statistical significance is represented as follows: * p < 0.05, ** p < 0.01, *** p < 0.001. PRL, prolactin; IGFBP1, insulin-like growth factor binding protein 1.
downregulated in human decidual tissues of SPE compared to those of NP group. In the in vitro culture system, the mRNA expressions of PGK1, PGK1P2 and the protein level of PGK1 increased significantly during decidualization and this augment was time dependent. When PGK1 and PGK1P2 were knocked down, the levels of PRL and IGFBP1 decreased sharply. More importantly, we found that PGK1P2 acted as a ceRNA to regulate PGK1 expression by acting as a sponge for miR-330-5p during human decidualization process. These findings demonstrate PGK1 and PGK1P2, a pair of ceRNAs linked by miR-330-5p, participate in human decidualization, and their deficiency in human decidua is associated with SPE. Lots of evidences have showed that regulatory modalities jointly exerted by LncRNAs and pseudogenes play vital role in human diseases. LncRNA is remarkable for its functional heterogeneity and can bind miRNA or a miRNA’s target sequence. Pseudogenes are copies of protein-coding genes and share a high sequence similarity with their parental genes which generate the potential for sequence-specific regulation [16]. Hence, a ceRNA hypothesis has been proposed. In this hypothesis, RNAs can crosstalk with each other by competing for shared miRNAs [17]. miRNAs, being endogenous small RNAs of 19–25 nucleotides, can regulate gene expression by destabilizing/ degrading mRNAs through imperfect base pairing to the “seed match” region in
induced to decidualization for 4 days. QPCR analysis revealed that miR330-5p suppressed the mRNA abundance of both PGK1P2 and PGK1, the mRNA level of PRL and IGFBP1 decreased significantly at the same time (Fig. 5A and D). In addition, the PGK1 protein level showed the same patterns by western blot analysis shown in Fig. 5B, C, E and F. To further test whether the expression level of PGK1P2 and PGK1 were linked by miR-330-5p, miR-330-5p inhibitor was transfected into hESCs and primary ESCs, the cells were then induced to decidualization for 4 days and 3 days respectively. QPCR analyses indicated that the transfection of miR-330-5p inhibitor not only increased PGK1P2 level but also increased PGK1 level in both hESCs and primary ESCs. Meanwhile the PRL and IGFBP1 mRNA level increased significantly (Fig. 6A and D). Moreover, the PGK1 protein abundance also increased as shown in Fig. 6B, C, E and F.
4. Discussion In this study, we focused on the roles of PGK1 and PGK1P2 in the decidualization of hESCs and primary ESCs and their relationship with SPE. We also explored the interrelation between PGK1 and PGK1P2 to extend our study. We demonstrated that PGK1 decreased significantly both at the RNA and protein levels, and PGK1P2 was significantly 42
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Fig. 5. Effects of miR-330-5p mimic transfection on in vitro decidualization of hESCs and primary ESCs. A–C are the experimental results of hESCs, D–F are the experimental results of primary ESCs. A, D The mRNA expression of PGK1, PGK1P2, PRL, IGFBP1 and miR-330-5p after miR-330-5p mimic transfection combined induced decidulization. B, E Bands representing PGK1 and β-actin proteins. C, F The relative expression levels of PGK1 compared to that of β-actin. The data are presented as the means ± SEM. Statistical significance is represented as follows: * p < 0.05, ** p < 0.01, *** p < 0.001. PRL, prolactin; IGFBP1, insulin-like growth factor binding protein 1.
remodeling consequently [3]. Disturbances of angiogenesis signal pathways have been proved to be associated with PE [33]. VEGF is famous as an angiogenesis factor in many tissues and its decreased expression has been implicated in the pathophysiology of PE [34,35]. Researches have demonstrated extracellular PGK1 in prostate cancer causes a reduction of plasmin which can facilitate the cleavage of plasminogen to generate the vascular inhibitor angiostatin, which is known as an important regulator of an “angiogenic switch” [13]. In our study, we revealed the expression of VEGF decreased markedly with the down-regulated expression of PGK1, also providing a potential risk for decidual impairment. But further investigation exploring the relationship between PGK1 and VEGF is still needed. Furthermore, previous researches have provided unequivocal proof that glycolysis is activated in decidua for supporting early pregnancy when decidualization starts [20,36]. Decidual cells exhibit a high rate of glycolysis because it is an important source of precursors for synthesis of amino acids and lipids [37,38] and can provide sufficient energy for cell proliferation fast. The glycolysis-related genes such as GLUT1 [39] and PDK4 [40] are found to be induced during human decidualization and the impairment of glycolysis in placenta is found to be related with PE pathogenesis [38]. Therefore, we speculated as a key enzyme in the glycolytic pathway the deficiency of PGK1 in decidua might jeopardize glycolysis and subsequently contribute the
the 5’ UTR, CDS, or 3’ UTR of the mRNA [24]. Accumulating lines of evidence have led to the view that miR-330-5p is involved in the progression of prostate cancer [25], colorectal cancer [26] and cutaneous malignant melanoma [27]. In the present study, we have demonstrated that PGK1P2 was a ceRNA for PGK1 against miR-330-5p. This ceRNAbased regulatory circuitry plays important roles in post-transcriptional regulation [28] and represents a new phenomenon in PE physiological and pathological processes. In humans, decidualization is a response to progesterone during the secretory phase of menstrual cycle independent of the presence of an implanting blastocyst. At that time, ESCs transform and differentiate into decidualized stromal cells; decidual markers, such as PRL and IGFBP1, accumulate markedly [4]. As a consequence, decidualized stomal cells acquire the special ability to regulate trophoblast invasion and to resist inflammatory and oxidative abuse [29]. Obviously, decidualization is the preparation of the “maternal soil” for the “embryo seed” and is vital for embryo implantation and pregnancy maintenance [30]. The decidualization reaction is initiated around blood vessels [31]. It can promote maternal spiral arteries remodelling into high-capacitance vessels accompanied by enhanced expression of key angiogenic factors and microvascular angiogenesis [32]. The abnormalities of decidualization could compromise trophoblast invasion and spiral artery 43
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Fig. 6. Effects of miR-330-5p inhibitor transfection on in vitro decidualization of hESCs and primary ESCs. A–C are the experimental results of hESCs, D–F are the experimental results of primary ESCs. A, D The mRNA expression of PGK1, PGK1P2, PRL, IGFBP1 and miR-330-5p after miR-330-5p inhibitor transfection combined induced decidulization. B, E Bands representing PGK1 and β-actin proteins. C, F The relative expression levels of PGK1 compared to that of β-actin. The data are presented as the means ± SEM. Statistical significance is represented as follows: * p < 0.05, ** p < 0.01, *** p < 0.001. PRL, prolactin; IGFBP1, insulin-like growth factor binding protein 1.
Appendix A. Supplementary data
pathogenesis of PE. In summary, we speculate that PGK1 and PGK1P2 are a pair of ceRNAs against miR-330-5p and they play a vital role in human decidualization by regulating glycolysis metabolism and angiogenesis. The deficiency of PGK1 and PGK1P2 in the decidua jeopardizes the decidualization process, which subsequently leads to the occurrence of PE.
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Acknowledgements The authors would like to thank all the patients and their relatives for their participation during this study. This work was supported by the National Natural Science Foundation of China (NSFC: 31471399 and 31671199) and National Key R&D Program of China (2017YFC1001403) to C. Z.; by a grant from the Major Program of the National Natural Science Foundation of China (81490743) to Z.-J. C. and by the Shanghai Commission of Science and Technology (17DZ2271100).
Conflicts of interest There are no conflicts of interest. 44
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