LIF–STAT signaling in decidual cells: a possible role in embryo implantation and early pregnancy

In this study, we investigate the effects of miRNA-138-5p and probable G-protein coupled receptor 124 (GPR124)-regulated inflammasome and downstream leukemia inhibitory factor (LIF)–STAT and adhesion molecule signaling in human decidual stromal cells. After informed consent was obtained from women aged 25–38 years undergoing surgical termination of the normal pregnancy and spontaneous miscarriage after 6–9 weeks of gestation, human decidual stromal cells were extracted from the decidual tissue. Extracellular vesicles (EVs) with microRNA (miRNA) between cells have been regarded as critical factors for embryo–maternal interactions on embryo implantation and programming of human pregnancy. MicroRNA-138-5p acts as the transcriptional regulator of GPR124 and the mediator of downstream inflammasome. LIF-regulated STAT activation and expression of integrins might influence embryo implantation. Hence, a better understanding of LIF–STAT and adhesion molecule signaling would elucidate the mechanism of microRNA-138-5p- and GPR124-regulated inflammasome activation on embryo implantation and pregnancy. Our results show that microRNA-138-5p, purified from the EVs of decidual stromal cells, inhibits the expression of GPR124 and the inflammasome, and activates the expression of LIF–STAT and adhesion molecules in human decidual stromal cells. Additionally, the knockdown of GPR124 and NLRP3 through siRNA increases the expression of LIF–STAT and adhesion molecules. The findings of this study help us gain a better understanding the role of EVs, microRNA-138-5p, GPR124, inflammasomes, LIF–STAT, and adhesion molecules in embryo implantation and programming of human pregnancy.


Introduction
The functional cross talk between embryos and maternal endometrium has a critical role in successful embryo implantation, placentation, and subsequent pregnancy (Gellersen et al. 2010, Teklenburg et al. 2010, Wu et al. 2015).Due to the rejection of the maternal immune system in the process of embryo implantation (Blois et al. 2007), the specific embryo-maternal interactions through active intracellular and released protein changes in the peri-implantation phase are shown to promote successful formation of pregnancy (Greening et al. 2016).The role of extracellular vesicles (EVs) and microRNAs (miRNA) in decidualization for embryo-maternal interactions of decidual stromal cells has now been investigated in the process of embryo implantation (Wu et al. 2022).miRNAs may contribute to inflammation and vascular alteration associated with other mechanisms, such as embryonal development or the tissue remodeling and integrity of the angiogenesis, still needs to be investigated.Leukemia inhibitory factor (LIF) is one growth factor that controls multiple biological functions.During the process of embryo implantation, LIF reveals its expression and actions on the embryos and endometrium (Cullinan et al. 1996, Fitzgerald et al. 2008).Under in vitro conditions, LIF might modulate embryo-endometrial interactions by activating STAT3 and integrins to enhance embryo implantation (Ceydeli et al. 2006, Suman et al. 2013).Therefore, an extensive investigation of LIF-STAT and integrin signaling may improve the outcome of embryo implantation and subsequent pregnancy.
In this study, we have tried to identify extracted EVs and miRNAs from decidua and decidual stromal cells.miRNArelated inflammasomes and subsequent mechanisms of angiogenesis and tissue remodeling are still not well known.Our study aimed to identify new EV-related miRNAs involved in embryo implantation and pregnancy.We investigated the downregulation of miR-138-5p through the target gene of miR-138a-5p and downstream signaling.The NLRP3 inflammasome is considered important in immunity and human pathophysiology, but the mechanism of microRNA-138-5p-regulated NLRP3 inflammasome and downstream signaling are yet to be elucidated.In the current study, we investigate the roles of miRNA-138-5p, probable G-protein coupled receptor 124 (GPR124), NLRP3 inflammasome, and subsequent LIF-STAT and integrin signaling in embryo implantation and early pregnancy.

Cell culture
To explore the effects of miRNA-138-5p and GPR124regulated inflammasome and downstream LIF-STAT signaling in human decidual stromal cells, after obtaining informed consent from women aged 25-38 years undergoing surgical termination of normal pregnancy and spontaneous miscarriage after 6-9 weeks of gestation, human decidual stromal cells were extracted from the decidual tissue.The involvement of human subjects in this study was approved by the Institutional Review Board of Chang Gung Memorial Hospital (CGMH-IRB nos. 201601676A3, 201702112B0, 201802242A3, 201902015B0, 202002376B0, and 202100385B0).Enzymatic digestion and mechanical dissociation were applied to purify human decidual stromal cells from the decidual tissue according to a modified protocol (Chou et al. 2003).Briefly, the human decidual tissue was minced and treated with type IV collagenase (Sigma-Aldrich) and DNase type I in a shaking water bath at 37°C for 90 min.The cell digest was then passed through a 70 μm filter.The decidual epithelial and stromal cells were collected in a 50 mL tube.Next, decidual epithelial cells were separated from decidual stromal with a 45 μm filter.The decidual stromal cells were subsequently pelleted by centrifugation at 1000 g for 5 min at room temperature.The cell pellet was washed once, resuspended, and plated in Dulbecco's modified Eagle's medium (DMEM) containing 25 mM glucose, l-glutamine, antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin), and supplemented with 10% fetal bovine serum (FBS).

Extracellular vesicle isolation
For isolation of EVs, the primary decidual stromal cells were cultured in DMEM, including 25 mM glucose, 200 mM l-glutamine, and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin), and supplemented with 10% FBS at 37°C, 5% CO 2 , following which the medium was converted to DMEM supplemented with Gibco™ extracellular vesicle-depleted FBS (Thermo Fisher, #A2720801) for 72 hours, and the supernatants were collected for isolation of EVs.Then, the culture medium was centrifuged at 1200 g for 10 min to remove impure cells and filtration through 0.22 μm filters to remove cell debris and particles larger than 200 nm, then centrifuged twice at 10,000 g for 30 min to remove larger microvesicles (MVs).EVs were pelleted by ultracentrifugation at 100,000 g for 60 min (Optima XE-90, Beckman Coulter, rotor Ti SW28) (all steps were performed at 4°C).The EVs were dissolved in 100 μL of PBS solution to prepare a suspension, which was preserved at −80°C.

Extracellular vesicle RNA isolation
TRI Reagent LS (750 μL) (Sigma, #T3934) was added to 250 µL EV sample.Subsequently, 200 µL of chloroform were added to each sample, mixed thoroughly by shaking for over 30 s, and incubated at room temperature for 10 min.Phase separation was performed by centrifugation at 12,000 g at 4°C for 15 min.The upper aqueous phase was collected.A volume of 1 µL glycogen solution (20 μg/μL) and an equal volume of isopropanol were added to each sample for RNA precipitation.Samples were mixed, incubated 1.5 h at −80°C, and then centrifuged at 12,000 g at 4°C for 30 min to pellet RNAs.The pellet was washed with 75% ethanol and centrifuged at 12,000 g at 4°C for 10 min.Pellets were dried for 10 min before resuspending in 15 μL diethyl pyrocarbonate (DEPC)-treated water, and purified RNA was quantified using NanoDrop.Total extracellular vesicle RNAs were reverse transcribed to cDNAs using TaqMan™ MicroRNA Reverse Transcription Kit according to the manufacturer's instructions.Quantitative PCR was performed using TaqMan™ MicroRNA assay in Applied Biosystems™ QuantStudio™ 5 Real-Time PCR System.All TaqMan™ MicroRNA assays (hsa-miR-138-5P: 0022084; U6 snRNA: 001973) were purchased from Applied Biosystems.U6 snRNA served as the internal control for miRNAs.Quantitative PCR conditions were: predenaturation at 95°C, 10 min followed by 95°C 15 s, 60°C 60 s, and totally 40 cycles to detect the expression of miR-138-5P and U6 snRNA.Expression levels are triplicated as the cycle threshold (Ct) value of the candidate gene relative to the Ct value of the housekeeping gene.Fold changes in the expression of each miRNA were calculated by a comparative threshold cycle (Ct) method using the formulas:

Nanoparticle tracking analysis
The isolated EVs were evaluated using NanoSight NS300 (Malvern Instruments, Worcestershire, UK) equipped with a blue laser (488 nm).The laser illuminated the nanoparticles, and their Brownian motion was captured for 60 s.The video was subjected to nanoparticle tracking analysis (NTA) using the NanoSight particle tracking software to calculate nanoparticle concentrations and size distribution.

Extracellular vesicle visualization by transmission electron microscopy
The sample was fixed with 2.5% glutaraldehyde for 30 min at room temperature.Extracellular vesicle suspension of 10-20 μL was absorbed onto formvar carbon-coated copper electron microscopy grids (300 mesh) at room temperature for 5 min and then subjected to 3% uranyl acetate staining for 30 min.Grids were washed three times with PBS and were maintained in a semi-dry state before observation by TEM (JEOL JEM-1400, Tokyo, Japan).

Reverse transcription quantitative polymerase chain reaction
Total extracellular vesicle RNAs were reverse transcribed to cDNAs using TaqMan™ MicroRNA Reverse Transcription Kit (Applied Biosystems PN: 4366596) with primers for miR-138-5p and U6 small nuclear RNA (hsa-miR-138-5p (assay ID: 002284); U6 snRNA (assay ID: 001973), Applied Biosystems) according to the manufacturer's instructions.Quantitative PCR was performed using QuantStudio™ 5 Real-Time PCR System.Real-time PCR reactions with cDNAs were performed in a 20 μL final volume.A reaction mix containing 2 μL cDNAs, 10 μL 2 × TaqMan® Universal PCR MasterMix II, no UNG (#4440040) and 1 μL 1 × TaqMan™ MicroRNA assay (20×; #4427975), and 7 μL DEPC-treated water was loaded into each well.All of TaqMan™ MicroRNA assays (hsa-miR-138-5p (assay ID: 002284); U6 snRNA (assay ID: 001973), Applied Biosystems) were purchased from Life Technologies.U6 snRNA served as the internal control for miRNAs.Quantitative PCR conditions were: pre-denaturation at 95°C, 10 min followed by 40 cycles of 95°C for 15 s, and 60°C for 1 min to detect the expression of miR-138-5P and U6 snRNA.Expression levels are triplicated as cycle threshold (Ct) value of the candidate gene relative to the Ct value of the housekeeping gene.Fold changes in expression of each miRNA were calculated by a comparative threshold cycle (Ct) method using the formulas:

Small interfering RNA transfection
SiCONTROL NON-TARGETING pool siRNA, siGENOME ON-TARGETplus SMARTpool human GPR124 siRNA, and NLRP3 siRNA were purchased from Dharmacon.The cells were transfected with siRNA (50 nM) using Lipofectamine RNAiMAX.After a 48 h transfection, the medium was removed and changed to fresh serumfree medium.To examine the siRNA transfection, cells were transfected with 50 nM si-GLO (Dharmacon) for 48 h.The transfection efficiency was examined by fluorescent microscopy.

Protein quantification assays
Cell culture supernatants were analyzed, at the prescribed times, for the existence of protein activity by using mouse GPR124, NLRP3, ASC, LIF, IL-1β, IL-18, pSTAT3, STAT3, and intergrin β DuoSet ELISA (R&D and MBL) following the manufacturer's instructions.ATP was quantified in cell supernatants using ATP Determination Kit (Life Technologies).

Statistical analysis
The results are expressed as the means ± s.e.m.Statistical evaluation was executed with the t-test for paired data.Multiple comparisons were first analyzed by one-way ANOVA, followed by Tukey's multiple comparison tests.A P-value of <0.05 was considered statistically significant.

GPR124-regulated expression of IL-18, IL-1β, NLRP3 inflammasome, and LIF in endometrial decidual stromal cells
Endometrial decidual stromal cells were transfected with control siRNA (si-ctrl) and GPR124 siRNA (si-GPR124) for 48 h.The decidual stromal cells and culture medium were collected at 48 h after transfection to examine the expression of IL-18, IL-1β, GPR124, NLRP3, and LIF by immunoblot and ELISA analyses.Pretreatment with GPR124 siRNA abolished the expressions of GPR124, NLRP3, IL-18, and IL-1β in endometrial decidual stromal cells, as shown in Fig. 3A and B. Meanwhile, LIF expression was promoted by GPR124 siRNA pretreatment in decidual stromal cells (Fig. 3A and B).

NLRP3-regulated expression of IL-18, NLRP3, IL-1β inflammasome, and LIF in endometrial decidual stromal cells
Endometrial decidual stromal cells were transfected with control siRNA (si-ctrl) and NLRP3 siRNA (si-NLRP3) for 48 h.The decidual stromal cells and culture medium were collected at 48 h after transfection to examine the expression of IL-18, NLRP3, IL-1β, and LIF by immunoblot and ELISA analyses.Pretreatment with NLRP3 siRNA weakened the expressions of IL-18, NLRP3, and IL-1β in endometrial decidual stromal cells (Fig. 4A and B).Furthermore, the protein expression of LIF was enhanced by NLRP3 siRNA pretreatment in decidual stromal cells (Fig. 4A and B).

Impacts of miRNA-138-5p on GPR124, LIF, p-STAT3, and STAT3 on human decidual stromal cells
Human decidual stromal cells were transfected with miR-138-5P (50 nM) for 48 h.The decidual stromal cells and culture medium were collected at 48 h after transfection to examine the expression of GPR124, LIF, p-STAT3, and STAT3 by immunoblot and ELISA analyses.Pretreatment with miR-138-5P weakened the expressions of GPR124 in endometrial decidual stromal cells, as shown in Fig. 5A and B.Moreover, the protein expressions of LIF, p-STAT3, and STAT3 were promoted by miR-138-5P pretreatment in decidual stromal cells, as shown in Fig. 5A and B.

GPR124-regulated expression of GPR124, LIF, p-STAT3, STAT3 and intergrin β in endometrial decidual stromal cells
Endometrial decidual stromal cells were transfected with control siRNA (si-ctrl) and GPR124 siRNA (si-GPR124) for 48 h.The decidual stromal cells and culture medium were collected at 48 h after transfection to examine the expression of GPR124, LIF, p-STAT3, STAT3, and intergrin β by immunoblot and ELISA analyses.Pretreatment with GPR124 siRNA weakened the expressions of GPR124 in endometrial decidual stromal cells, as shown in Fig. 6A and B.Moreover, the protein expressions of LIF, p-STAT3, STAT3, and integrin β were increased by GPR124 siRNA pretreatment in decidual stromal cells, as shown in Fig. 6A and B.

NLRP3-regulated expression of NLRP3, LIF, p-STAT3, STAT3, and intergrin β in endometrial decidual stromal cells
Endometrial decidual stromal cells were transfected with NLRP3 siRNA (si-NLRP3) and control siRNA (si-ctrl) for 48 h.The decidual stromal cells and culture medium were collected at 48 h after transfection to examine the expression of NLRP3, LIF, p-STAT3, STAT3, and intergrin β by immunoblot and ELISA analyses.Pretreatment with NLRP3 siRNA abolished the expressions of NLRP3 in endometrial decidual stromal cells, as shown in Fig. 7A and B. Meanwhile, the protein expressions of LIF, p-STAT3, STAT3, and integrin β were promoted by NLRP3 siRNA pretreatment in decidual stromal cells, as shown in Fig. 7A and B.

Discussion
Collectively, our findings demonstrated that the purified EVs from decidual stromal cells, the microRNA-138-5p-inhibited GPR124 and inflammasome expression, and microRNA-138-5p activated the expression of LIF-STAT and adhesion molecules in human decidual stromal cells.Additionally, the knockdown of GPR124 and NLRP3 through siRNA increases the expression of LIF-STAT and adhesion molecules (Fig. 8).The embryomaternal cross talk during embryo implantation and early pregnancy plays an essential role in the control of human reproduction.Many protocols have been created to induce decidualization; however, the period of challenge varies markedly between studies, ranging from a couple of days to 10 or more days (Gellersen & Brosens 2014).The heterogeneity in experimental design with the diverse extent of cellular reprogramming upon decidualization (Gellersen & Brosens 2003) is the rationale for designing this study to be conducted using human decidual stromal cells collected from the decidual tissue.EVs and cargo of EVs between cells have been considered critical factors for embryo implantation and programming of human pregnancy.This study aimed to evaluate the functional embryomaternal cross talk during the embryo implantation and placentation through EV-carried microRNA-138-5p, downstream GPR124-regulated inflammasome, LIF-STAT, and adhesion molecules signaling in human decidual stromal cells.LIF is a member of the IL-6 cytokine family.The biological role of the LIF in reproduction has been characterized previously, including blastocyst development, initiating embryouterine communication, implantation, and trophoblast invasion (Nicola & Babon 2015).The expression of LIF was demonstrated in the endometrium in fertile and infertile women, while a differential expression of LIF was observed in women with implantation failure and infertility (Wu et al. 2013, Zare et al. 2020).EVs are involved in the complex biological processes of cell-cell interactions, including embryo-endometrial interactions.The present evidence demonstrates that EVs integrate into the target cell's plasma membrane and secrete their cargo, such as miRNAs, into the cell, where they can instantly interact with the target cell (Thery et al. 2002, Morelli et al. 2004).Our findings showed that extracted EVs from decidua and decidual stromal cells were investigated by immunoblot analysis, TEM, and nanoparticle tracking analysis.These EVs may contain miRNAs that can contribute to embryo implantation and pregnancy.miRNAs, a flock of non-coding RNAs, play a role in controlling the expression of target genes by partly or wholly binding to their independent complementary sequences (Zammar et al. 2014, Araldi & Suarez 2016).The expression profiles of miRNAs in embryo implantation and early pregnancy may have a role in some pathological regulation, possibly specific to spontaneous miscarriage.The impacts of some miRNAs in specific actions concerning the pathophysiology of spontaneous miscarriage have been shown in several studies with different mechanisms, such as reducing the proliferation and migration of endothelial cells and smooth muscle cells (Ferreira et al. 2014, Zammar et al. 2014, Huang et al. 2017).The contribution of miRNAs in regulating underlying mechanisms associated with abnormal embryo implantation and pregnancy needs to be well investigated.In our previous study (Wu et al. 2022), we demonstrated differential expression of miRNAs between normal pregnancy and spontaneous miscarriage in a hierarchical clustering analysis by microarray analysis of miRNAs in the endometrial tissue, where we showed that miRNA-138-5p is downregulated in spontaneous miscarriage compared to normal pregnancy.MicroRNA-138-5p not only has a vital role in the brain but also in peripheral tissues (Zottel et al. 2020).Furthermore, gene ontology analysis showed that microRNA-138-5p promotes physiological actions such as angiogenesis, cell migration, and placenta establishment, suggesting that the malfunction in these physiological actions contributes to abnormal embryo implantation and pregnancy (Lala & Nandi 2016, Salomon et al. 2017).In our previous study (Wu et al. 2022), we searched the target genes of miR-138-5p by using established miRNA target prediction programs and identified GPR124 as a potential target of miR-138-5p using TargetScan.GPR124 is an orphan G protein-coupled receptor that functionally modulates physiological angiogenesis and embryonic alteration within the central nervous system (Erkinova et al. 2018, Florian et al. 2020).The physiological role of GPR124 is established in angiogenesis and atherosclerosis potentially associated with embryo implantation.Our previous results demonstrated an opposite interaction between the expression of miR-138-5p and GPR124 in endometrial tissues (Wu et al. 2022).Our previous results propose that the interaction of miR-138-5p and GPR124 results in embryo implantation and early pregnancy; however, the underlying mechanism of this interaction still needs to be investigated.
Inflammasomes trigger inflammatory actions, which subsequently activate pro-inflammatory cytokines IL-1β and IL-18, and intercede cytokine production and programmed cell fate (Liu et al. 2016, Shi et al. 2017).Here, we showed the increased expression of NLRP3 inflammasome, IL-18, and IL-1β in spontaneous miscarriage compared to normal pregnancy; meanwhile, the expressions of IL-18, NLRP3, and IL-1β are modulated by miR138-5p and GRP124 in decidual stromal cells, indicating that IL-18 inflammasome, NLRP3, and IL-1β could be related to abnormal pregnancy.Furthermore, in the present study, we demonstrate for the first time that miR138-5p and GRP124 may participate in early pregnancy by mediating the expression of NLRP3 inflammasome, IL-18, and IL-1β, exploring the complex process of embryo implantation and early pregnancy.
Based on the evidence of this study, the challenge of human decidual stromal cells with microRNA138-5p leads to significantly increased activation of LIF, p-STAT3, STAT3, and integrin β; meanwhile, reduced expression of NLRP3, GPR124, IL-1β, and IL-18.Our results indicate that miRNA138-5p directly controls the expression of LIF, p-STAT3, STAT3, NLRP3, integrin β, GPR124, IL-1β, and IL-18 in human decidual stromal cells.At the same time, in this study, GPR124 siRNA was applied to specifically knock down the protein expression of GPR124 in human decidual stromal cells.Contrary to GPR124 with siRNA weakened the GPR124-modulated activation of NLRP3, IL-18, IL-1β, The proposed signaling pathways involved in the leukaemia inhibitory factor-STAT signaling and adhesion molecules in embryo implantation and early pregnancy.These results reveal the vital function of microRNA138-5p and GPR124 in modulating embryo implantation and early pregnancy through the mediation of LIF-STAT signaling and adhesion molecules and the subsequent activation of NLRP3 inflammasome, IL-18, and IL-1β signaling.

Figure 1
Figure 1Extracellular vesicles in decidual stromal cells by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and immunoblot analysis.(A) Laser illuminated the nanoparticles, and their movement under Brownian motion was captured for 60 s.The video was subjected to NTA using the NanoSight particle tracking software to calculate nanoparticle concentrations and size distribution.The black line is the average of all data.Green shades are s.d. of three measurements.Blue numbers mean the size (nm) of extracellular vesicles.(B) Isolated extracellular vesicles derived from decidual stromal cells by ultracentrifugation visualized in TEM.In TEM image, EVs (arrow) displayed a round morphology with a lipid bilayer structure.(C) The extracellular vesicle supernatant was denatured in 4× sodium dodecyl sulfate (SDS) buffer and subjected to western blot analysis (10% SDS-PAGE; 50 μg protein/lane) using rabbit CD63, CD9, CD81, and TSG101 antibodies.The control sample contained DSC cell lysate and the exosome sample contained purified exosomes.Beta-actin served as a control.DSC, decidual stromal cells.
Figure 2 Effects of miRNA-138-5p on GPR124, NLRP3, IL-18, IL-1β inflammasome, and LIF on human decidual stromal cells.Human decidual stromal cells were transfected with miR-138-5P or miR-NC (control) (50 nM) for 48 h.The decidual stromal cells and the culture medium were collected at 48 h after transfection to examine the expression of GPR124, NLRP3, IL-18, IL-1β, and LIF.(A) Immunoblot analysis for protein expression in decidual stromal cells.Expression levels quantified by densitometry.IL-18 and IL-1β in the chart are mature forms that have been quantified.(B) ELISA detection of protein concentration in collected culture medium.All expression results shown as mean ± s.e.m. of three independent experiments, normalized to miR-NC expression, and differences assessed by paired t-tests (*P < 0.05 , versus control).DSC, decidual stromal cells.

Figure 3 GPR124
Figure 3 GPR124-regulated expression of NLRP3, IL-18, IL-1β inflammasome, and LIF in endometrial decidual stromal cells.Endometrial decidual stromal cells were transfected with control siRNA (si-ctrl) or GPR124 siRNA (si-GPR124) (50 nM) for 48 h.Decidual stromal cells and the culture medium were collected 48 h after transfection to examine protein expression of GPR124, NLRP3, IL-18, IL-1β, ASC, and LIF.(A) Immunoblot analysis for protein expression in decidual stromal cells.Expression levels quantified by densitometry.IL-18 and IL-1β in the chart are mature forms that have been quantified.(B) ELISA detection of protein concentration in the culture medium.Expression results are shown as mean ± s.e.m. of three independent experiments, normalized to si-ctrl, and differences assessed by paired t-tests.DSC, decidual stromal cells.

Figure 4 NLRP3
Figure 4 NLRP3-regulated expression of NLRP3, IL-18, IL-1β inflammasome, and LIF in endometrial decidual stromal cells.Endometrial decidual stromal cells were transfected with control siRNA (si-ctrl) and NLRP3 siRNA (si-NLRP3) (50 nM) for 48 h.Decidual stromal cells and the culture medium were collected 48 h after transfection to examine the expression of NLRP3, IL-18, IL-1β, and LIF.(A) Immunoblot analysis for protein expression in decidual stromal cells.Expression levels quantified by densitometry.IL-18 and IL-1β in the chart are mature forms that have been quantified.(B) ELISA detection of protein concentration in the culture medium.Expression results shown as mean ± s.e.m. of three independent experiments, normalized to si-ctrl, and differences assessed by paired t-tests.DSC, decidual stromal cells.

Figure 5
Figure 5Impacts of miRNA-138-5p on GPR124, LIF, p-STAT3, and STAT3 on human decidual stromal cells.Human decidual stromal cells were transfected with miR-138-5p or miR-NC (50 nM) for 48 h.Decidual stromal cells and the culture medium were collected 48 h after transfection to examine the expression of GPR124, LIF, p-STAT3, and STAT3.(A) Immunoblot analysis for protein expression in decidual stromal cells.Expression levels quantified by densitometry.(B) ELISA detection of protein concentration in culture medium.Expression results shown as mean ± s.e.m. of three independent experiments, normalized to si-ctrl, and differences assessed by paired t-tests.DSC, decidual stromal cells.

Figure 6 GPR124
Figure 6 GPR124-regulated expression of GPR124, LIF, p-STAT3, STAT3, and integrin β in endometrial decidual stromal cells.Endometrial decidual stromal cells were transfected with control siRNA (si-ctrl) or GPR124 siRNA (si-GPR124) (50 nM) for 48 h.Decidual stromal cells and the culture medium were collected 48 h after transfection to examine the expression of GPR124, LIF, p-STAT3, STAT3, and integrin β. (A) Immunoblot analysis for protein expression in decidual stromal cells.Expression levels were quantified by densitometry.(B) ELISA detection of protein concentration in culture medium.Expression results shown as mean ± s.e.m. of three independent experiments, normalized to si-ctrl, and differences assessed by paired t-tests.DSC, decidual stromal cells.

Figure 7 NLRP3
Figure 7 NLRP3-regulated expression of NLRP3, LIF, p-STAT3, STAT3, and integrin β in endometrial decidual stromal cells.Endometrial decidual stromal cells were transfected with control siRNA (si-ctrl) and NLRP3 siRNA (si-NLRP3) (50nM) for 48 h.Decidual stromal cells and the culture medium were collected 48 h after transfection to examine the expression of NLRP3, LIF, p-STAT3, STAT3, and integrin β. (A) Immunoblot analysis for protein expression in decidual stromal cells.Expression levels quantified by densitometry.(B) ELISA detection of protein concentration in culture medium.Expression results shown as mean ± s.e.m. of three independent experiments, normalized to si-ctrl, and differences assessed by paired t-tests.DSC, decidual stromal cells.