Chemerin-Induced Down-Regulation of Placenta-Derived Exosomal miR-140-3p and miR-574-3p Promotes Umbilical Vein Endothelial Cells Proliferation, Migration, and Tube Formation in Gestational Diabetes Mellitus

Gestational diabetes mellitus (GDM) leads to poor pregnancy outcomes and fetoplacental endothelial dysfunction; however, the underlying mechanisms remain unknown. This study aimed to investigate the effect of placenta-derived exosomal miRNAs on fetoplacental endothelial dysfunction in GDM, as well as to further explore the role of chemerin to this end. Placenta-derived exosomal miR-140-3p and miR-574-3p expression (next-generation sequencing, quantitative real-time PCR), its interactions with cell function (Cell Counting Kit-8, Transwell, tube formation assay), chemerin interactions (Western blotting), and placental inflammation (immunofluorescence staining, enzyme-linked immunosorbent assay) were investigated. Placenta-derived exosomal miR-140-3p and miR-574-3p were downregulated in GDM. Additionally, miR-140-3p and miR-574-3p inhibited the proliferation, migration, and tube formation ability of umbilical vein endothelial cells by targeting vascular endothelial growth factor. Interestingly, miR-140-3p and miR-574-3p expression levels were negatively correlated with chemerin, which induced placental inflammation through the recruitment of macrophage cells and release of IL-18 and IL-1β. These findings indicate that chemerin reduces placenta-derived exosomal miR-140-3p and miR-574-3p levels by inducing placental inflammation, thereby promoting the proliferation, migration, and tube formation of umbilical vein endothelial cells in GDM, providing a novel perspective on the underlying pathogenesis and therapeutic targets for GDM and its offspring complications.


Introduction
Gestational diabetes mellitus (GDM) is characterized by glucose intolerance at the onset of pregnancy, which is commonly diagnosed in the second or third trimester of pregnancy and is not clearly type 1 or 2 diabetes. GDM affects approximately 15% of all pregnancies worldwide and its incidence is increasing. Notably, GDM confers an increased risk of severe pregnancy complications, including macrosomia, shoulder dystocia, and neonatal hypoglycemia, along with an increased likelihood of long-term offspring metabolic diseases such as type 2 diabetes, obesity, and cardiovascular disease [1][2][3]. Therefore, it is crucial that the underlying molecular mechanisms, prognostic markers, and molecular therapeutic targets be identified to prevent GDM and improve the treatment outcomes for GDM complications.
Recent studies indicate that GDM is associated with fetoplacental endothelial dysfunction [4,5]. The fetoplacental vasculature plays a key role in trans-placental nutrient from the mother to the fetus; changes may lead to abnormal fetal growth and development, and even to alterations in the health status of the newborn, child, and adult. Accordingly, fetoplacental endothelial dysfunction in GDM may contribute to offspring complications; however, its underlying mechanisms remain poorly understood. Studies have shown that oxidative stress and inflammatory activation are related to cardiac and diabetic vascular endothelial dysfunction [6,7]. It has been reported that macrophage infiltration is increased in GDM placenta compared to the normal group [8]. Furthermore, the relative M1 macrophage ratio was higher in GDM placenta, while the M2 macrophage relative ratio is the opposite [9]. Additionally, several studies have documented changes to inflammatory cytokines such as interleukin-8 (IL-8), IL-15, and tumor necrosis factor α (TNF-α) in GDM placentas [10,11]. Placental inflammation has been observed in GDM, and may play a role in fetoplacental endothelial dysfunction [12].
Insulin, adenosine, and adipokine receptors are involved in the fetoplacental vascular dysfunction seen in GDM [13]. Chemerin, a recently discovered adipokine, plays a key role in the inflammatory response and metabolic disease [14]. Additionally, it has been shown that chemerin may affect angiogenesis by stimulating the production of vascular endothelial growth factor (VEGF) [15]. In women with preeclampsia, placental chemerin expression and release were increased, and chemerin overexpression inhibited the migration, invasion, and tube formation of human umbilical vein endothelial cells (HUVECs) [16]. Furthermore, chemerin stimulated M1 macrophage polarization and contributed to preeclampsia [17]. Previous studies have shown that levels of chemerin were higher in the blood, placental tissue, and cord blood of diabetic pregnancy patients [18,19]. Aggregation of chemerin led to macrophage recruitment and the release of inflammatory cytokines in the brains of offspring from chemerin-treated GDM dams [20]. However, whether chemerin has a role in regulating the function of fetoplacental endothelial cells in GDM has yet to be confirmed.
Extracellular vesicles (EVs) are nanosized particles containing bioactive content (including proteins, lipids and nucleic acids), and are enclosed by a lipid bilayer. The syncytiotrophoblast layer of the placenta secretes small EVs (exosomes and microvesicles) into the maternal circulation from as early as six weeks of gestation during pregnancy [21]. It has been shown that placental EVs are bioactive and contribute to the maternal proinflammatory state observed in GDM by stimulating HUVECs to release proinflammatory cytokines [22]. Moreover, human umbilical endothelium-derived exosomes have been reported to contribute to fetoplacental endothelial dysfunction in GDM pregnancies [23]. Exosomes are a subtype of extracellular vesicles that are specifically packaged with signaling molecules, including protein, messenger RNA (mRNA), and noncoding RNA. Exosome secretion has been reported in placental cells as a response to changes occurring across gestation. Placenta-derived exosomes regulate endothelial cell function and potentially affect the development of the maternal-fetal vasculature [24]. The concentration of exosomes from maternal plasma is significantly elevated in GDM, which promotes the release of proinflammatory cytokines from endothelial cells [22]. Extracellular vesicle-associated microRNAs (miRNAs) are altered in GDM in the context of insulin sensitivity [25]. Studies have shown that miRNAs mediate vascular endothelial dysfunction in chronic kidney disease, preeclampsia, and high-glucose conditions [26][27][28]. However, the impact of placentaderived exosomes and miRNAs on fetoplacental endothelial dysfunction in GDM is not well understood.
In this study, we analyze the effects of placenta-derived exosomal miRNAs on fetoplacental endothelial dysfunction in GDM and the role of chemerin. Placenta-derived exosomal miR-140-3p and miR-574-3p were reduced in chemerin-induced GDM, resulting in abnormal proliferation, migration, and tube formation of umbilical vein endothelial cells.

Patient Recruitment
Pregnant women with GDM and pregnant women with normal glucose levels who delivered by cesarean section were recruited to this study from the delivery suites of the Women's Hospital School of Medicine at Zhejiang University. GDM was diagnosed as abnormal blood glucose at any point according to the International Association of Diabetes and Pregnancy Study Group (IADPSG) criteria by 75g OGTT, including fasting plasma glucose (FPG) ≥5.1 mmol/L, 1h postprandial plasma glucose (PG) ≥10.0 mmol/L, and 2h-PG ≥8.5 mmol/L. Pregnant women who had experienced any of the following were excluded from the study: multiple pregnancies, assisted reproduction, an infection of the reproductive system during pregnancy, diabetes mellitus before pregnancy, or other pregnancy complications. The clinical characteristics are detailed in Table S1. This study was approved by the Ethics Committee of the Women's Hospital School of Medicine, Zhejiang University. All patients signed written informed consent forms.

Mouse Model Establishment
Male and female DBA/2J mice (aged 4-6 weeks) were purchased from Shanghai Jiesijie Laboratory Animals Co., Ltd. (Shanghai, China). All mice were acclimated for one week before treatment. Our experiments were approved by the Institutional Animal Research Committee and Ethics Committee of Zhejiang University. A typical diabetic model was established by intraperitoneal injection of streptozotocin (STZ, 40 mg/kg) for five consecutive days, beginning three days after pregnancy [29]. The chemerin-induced maternal diabetic model was established by intraperitoneal injection of chemerin (3-4 µg/g body weight, 2325-CM, RD, MN, USA) at gestational day (GD) 2, for three consecutive days [20], and the control group by intraperitoneal injection with normal saline at the same time. Both groups were verified by measuring fasting blood glucose (FBG) and fasting blood insulin levels (FBINS) at GD 10.5 and 18.5 using enzyme-linked immunosorbent assay (ELISA) kits (FBG, S0201S, Beyotime, Shanghai, China; FBINS, E-EL-M1382c, Elabscience, Wuhan, China) and the homeostasis model of assessing insulin resistance (HOMA-IR) ( Figure S1). HOMA-IR was calculated by (FBINS × FBG)/22.5.
Chemerin-induced maternal diabetic mice were divided randomly into two groups and compared to control mice. Chemerin treatment (chemR23 knockdown) was administered via an intravenous tail injection of 1 × 10 9 plaque-forming units (pfu) of chemR23short hairpin RNA (shRNA) lentivirus on GD 10.5. The controls and chemerin-induced diabetic mice were injected with a 1 × 10 9 pfu lentivirus vehicle. The mice were delivered by cesarean section on GD 18.5 and their placentas were collected for Western blotting and exosome isolation.

Preparation of Placenta-Derived Exosomes from Placenta Tissue and Exosomes from Trophoblast Cells
Placenta tissue was collected during delivery and stored in phosphate-buffered saline (PBS) at 4 • C. The large blood vessels and connective tissue of placental tissue were removed in an ultra-clean workbench. Then, placental tissue was rinsed repeatedly with PBS for several times. Villous tissue was cut into placental explants of 1-2 mm 3 size and rinsed repeatedly with 1640 medium (PM150110, Procell, Wuhan, China) with 10% fetal bovine serum (FBS, 10099141C, Gibco, Australia) for 3-5 times. Then, they were placed in a 12-well plate, to which was added 1640 medium containing 10% FBS. Placental explants were cultured in an incubator at 37 • C (5% CO2) for 48 h, and the culture medium was collected after 48 h. Exosome-enriched EVs were isolated by sequential centrifugation at 2000× g for 30 min at 4 • C and 10,000× g for 60 min. The pellets were then resuspended in PBS and washed twice, followed by filtration using a 0.22-mm filter. Exosome markers, including CD63, CD9, and CD81 [30], and a placenta-derived exosomal-specific marker (placental alkaline phosphatase, PLAP) were analyzed by Western blotting ( Figure S2A). Placenta-derived exosomes were characterized according to their morphology (typically a round or oval shape) and size (80-100 nm) using transmission electron microscopy (Titan, FEI) and nanoparticle tracking analysis (NTA, NanoSight, NS300, Malvern Panalytical, Malvern, UK) ( Figure S2B-D). After treating trophoblast cells with interleukin 18 (IL-18, 10 ng/mL, RAB0810, sigma, MO, USA), interleukin 1 beta (IL-1β, 10 ng/mL, HY-P7073, MCE, NJ, USA), or the supernatant from macrophage cells for 24 h, we collected the culture medium and isolated the exosomes.

Small RNA Sequencing
Next gene sequencing was performed according to the manufacturers' instructions from Illumina. Small RNA sequencing libraries were prepared using TruSeq Small RNA Sample Preparation Kits (RS-200-0012, Illumina, San Diego, CA, USA). After library preparation, the constructed libraries were sequenced using an Illumina Hiseq2000 with singleended 1X50bp read length.

Enzyme-Linked Immunosorbent Assay
For assessment of placental inflammation, placental tissue was removed from the pregnant women and mice through dissection and homogenized. The homogenates were analyzed using ELISA kits (IL-18, ml002294, mlbio, Shanghai, China; IL-1β, PI301, Beyotime). All ELISA measurements were performed according to the manufacturer's instructions.

Transwell Assay
A 200-µL aliquot of the elicited HUVECs or MUVECs (1 × 10 6 /mL) transfected with the hnRNP L vector or OE plasmid was transferred to the upper chambers in conjunction with placenta-derived exosome transfection with miR-140-3p and miR-574-3p agomir and/or antagomir into the lower chambers. The transwell chambers were taken out and washed with calcium-free PBS 24 h later, then fixed with 70% ethanol. After removing the non-migrating cells from the upper layer, the migrated cells were stained with 0.1% crystal violet and examined under a microscope. The number of cells stained with crystal violet was counted in five randomly selected fields at 400×.

Tube Formation Assay
HUVECs or MUVECs were seeded on growth factor-reduced Matrigel-coated 96-well plates at 3 × 10 4 cells per well for 24 h. Endothelial tubule formation was observed and photographed using an inverted microscope. Total tube lengths were measured in five randomly selected fields at 400x and then analyzed by Image J (National Institutes of Health, version 1.8.0).

Statistical Analysis
All statistical analyses were performed using IBM SPSS software (version 22.0; IBM Corp., Armonk, NY, USA). All data are presented as the mean ± standard deviation. Student's t-test was used to compare the differences between two groups, and analysis of variance was applied for comparison among multiple groups. Linear regression was performed between the observed therapeutic targets. A p-value < 0.05 was considered statistically significant.

Expression Levels of miR-140-3p and miR-574-3p Are Significantly Reduced in Placenta-Derived Exosomes from GDM
Our results revealed no difference in the concentration or number of placenta-derived exosomes collected from placental tissue between the normal (pregnancy) and GDM groups ( Figure S2C,D). Next-generation sequencing detected 38 medium-and high-abundance miRNAs with different expression levels in placenta-derived exosomes from GDM and normal pregnant women, including increased and decreased expression of 2 and of 36 miR-NAs, respectively ( Figure 1A and Table S2). We validated the data of seven miRNAs with medium and high abundance that showed the greatest changes using qRT-PCR. The expression levels of placenta-derived exosomal miR-140-3p and miR-574-3p, which showed the greatest decreases, were evaluated ( Figure 1C).  Placenta-derived exosomes from GDM and normal women were transfected with miRNA agomir and antagomir, respectively, to investigate whether exosomes affect endothelial cell function via miR-140-3p and miR-574-3p, including proliferation, migration,

Placenta-Derived Exosomes from GDM Altered Endothelial Cell Function Depending on miR-140-3p and miR-574-3p In Vitro
Placenta-derived exosomes from GDM and normal women were transfected with miRNA agomir and antagomir, respectively, to investigate whether exosomes affect endothelial cell function via miR-140-3p and miR-574-3p, including proliferation, migration, and tube formation. Figure 2A shows the changes in expression of miR-140-3p and miR-574-3p in response to successful transfection with their agomir and antagomir, respectively. No differences were detected in the uptake of placenta-derived exosomes by HUVECs among the groups ( Figure 2B). In contrast, the proliferation ( Figure 2C), migration ( Figure 2D), and total tube length ( Figure 2E) of endothelial cells were reduced after treatment of placenta-derived exosomes from GDM women transfected with miR-140-3p or miR-574-3p agomir, compared with the negative control, whereas the normal group showed an increase in these parameters after treatment of the placenta-derived exosomes transfected with their antagomir.

Chemerin Reduces Placenta-Derived Exosomal miR-140-3p and miR-574-3p Expression by Inducing Placental Inflammation
Chemerin is an adipokine and an important regulator of angiogenesis, inflammation, blood pressure, and the immune system. Studies have shown that chemerin from peripheral blood, adipose tissue, and placenta tissue is elevated in GDM [19,31]. In this study, we noticed that the expression levels of miR-140-3p and miR-574-3p in placentaderived exosomes were negatively correlated with chemerin in placental tissues in pregnant women ( Figure 3A). Additionally, we established a chemerin-induced diabetic mouse model and knocked down chemR23, which is the receptor of chemerin, via a chemR23 shRNA lentivirus injection. Figure 3B shows that chemR23 in placental tissue changes in response to successful knockdown of chemR23. We observed that the levels of miR-140-3p and miR-574-3p were decreased in placenta-derived exosomes from chemerin-induced GDM mice, and increased in GDM mice with chemR23 knocked down ( Figure 3C). This suggests that the reduced expression levels of exosomal miR-140-3p and miR-574-3p are associated with elevated chemerin in GDM.
Through immunofluorescence, we observed that macrophage infiltration was markedly exacerbated in GDM; co-localization of macrophages and chemerin was seen as well ( Figure 4A). Previous research has shown that the aggregation of chemerin in fetal brain tissue leads to cognitive impairment in GDM offspring; chemerin buildup induces macrophage recruitment and pyroptosis, which is characterized by the activation of caspase-1 and secretion of pro-inflammatory cytokines such as IL-1β and IL-18 [20,32]. In this study, elevated levels of IL-18 and IL-1β were observed in the placental tissue of pregnant women with GDM ( Figure 4B), which was observed in chemerin-induced diabetic mice as well; the expression levels of both decreased after knocking down chemR23 ( Figure 4C). Moreover, the changes observed in the NOD-like receptor family pyrin domain containing 3 (NRLP3), apoptosis-associated speck-like protein containing CARD (Asc), and cleaved caspase 1 expression in placental macrophages were consistent with IL-18 and IL-1β ( Figures 4D and S3). These data confirm that chemerin induces macrophage recruitment as well as the release of inflammatory factors in placental tissue.

MiR-140-3p and miR-574-3p Down-Regulate VEGF Expression in Endothelial Cells
Vascular endothelial growth factor (VEGF) is a diffusible endothelial cell-specific mitogen that mediates endothelial cell survival, migration, and proliferation during angiogenesis. Our results show that VEGF expression was significantly higher in the umbilical vein tissue of GDM patients ( Figure 5A) and was negatively associated with miR-140-3p and miR-574-3p levels in umbilical vein tissue ( Figure 5B). Therefore, we hypothesize that miR-140-3p and miR-574-3p affect endothelial cell function by regulating VEGF expression. miR-140-3p and miR-574-3p agomir reduced VEGF expression in vitro, while their antagomir had the opposite effect ( Figure 5C).
Through miRNAs target gene prediction software and protein interaction analysis, we found that miR-140-3p has multiple target genes and revealed more interactions between VEGF and other potential target genes ( Figure 5D). 3 -untranslated regions (3 UTRs) are the noncoding parts of mRNAs that regulate mRNA degradation, translation, and localization. We constructed wild-type and mutant luciferase reporter plasmids in the binding site in the 3'UTR of VEGF. miR-140-3p reduced the luciferase activity of wild-type VEGF 3'UTR, and had no effect on its mutant ( Figure 5E). miR-574-3p binds to heterogeneous nuclear ribonucleoprotein L (hnRNP L) and prevents hnRNP L from binding to the VEGFA mRNA, ultimately reducing VEGFA mRNA translation [33]. To clarify whether the mediating effect of miR-574-3p on the expression of VEGF depends on hnRNP L, an hnRNP L OE plasmid was constructed and hnRNP L and VEGF levels were determined by Western blotting. We observed an increase in hnRNP L OE plasmid levels and blockade of the inhibitory effect of miR-574-3p on VEGF expression ( Figure 5F). Moreover, OE of hnRNP L reversed the impairments in proliferation, migration, and tube formation caused by the miR-574-3p agomir ( Figure S5). Taken together, our results showed that miR-574-3p downregulates VEGF expression by competitively binding hnRNP L, thereby inhibiting the functional capacity of umbilical vein endothelial cells.  AG in MUVECs. A luciferase assay was applied to detect the luciferase activity (n = 3). (F) Protein levels of VEGF and hnRNP L after transfection with miRNA AG NC+vector and miR-574-3p AG+vector, miRNA AG NC+hnRNP L overexpression (OE), and miR-574-3p AG+hnRNPL OE were measured by Western blotting (n = 3). ** p < 0.01. ns: no significance.

Discussion
In this report, we have demonstrated that chemerin reduces placenta-derived exosomal miR-140-3p and miR-574-3p levels by inducing placental inflammation, thereby promoting the proliferation, migration, and tube formation of umbilical vein endothelial cells in GDM by upregulating VEGF expression.
GDM is a common metabolic disorder the prevalence of which is increasing in line with the increasing incidence of obesity and trend towards older maternal age seen worldwide. As an important mediator of the exchange of oxygen and nutrients between mother and fetus, the placenta plays an important role in fetal growth as well as the development of complications in offspring. Studies have found that placental abnormalities in GDM include an increased immature villous, increased placental weight, and increased measures of angiogenesis [34]. Fetoplacental endothelial dysfunction in GDM has been observed for decades, and plays a crucial role in offspring complications. However, the mechanism has not been elucidated to date. Studies have shown that fetoplacental endothelial dysfunction in GDM is related to the increased release of exosomes induced by hyperglycemia, hyperinsulinemia, and oxidative stress [23,35]. The concentration and bioactivity of placenta-derived exosomes present in maternal plasma were higher in GDM patients compared with those having a normal pregnancy, modulating the release of proinflammatory cytokines from endothelial cells [22]. Herein, we isolated placenta-derived exosomes from placenta tissue and found no difference in the concentration of placenta-derived exosomes between normal and GDM pregnant women. Placenta-derived exosomes from GDM women promoted the proliferation, migration, and tube formation of endothelial cells compared with those from normal women. Thus, fetoplacental endothelial dysfunction in GDM appears to be associated with changes in the bioactivity of placenta-derived exosomes. In addition, GDM along with failure of endothelial cells vasodilation since insulin resistance might result in blunted endothelium-derived NO-dependent vascular response to this hormone; this needs to be investigated further [36][37][38].
Placenta-derived exosomal miRNAs are released into the maternal circulation during pregnancy, and levels of a variety of miRNAs change in GDM patients [39]. Herein, we have shown an association of placenta-derived exosomal miRNA changes with the umbilical vein endothelial dysfunction seen in GDM. In addition, we have shown that placenta-derived exosomal miR-140-3p and miR-574-3p are significantly reduced in GDM patients. Previous studies on miR-140-3p and miR-574-3p have focused mainly on suppressing the proliferation, migration, and invasion of tumor cells [40][41][42][43]. Increased expression of miR-140-3p has been found to induce hypoxia reoxygenation-stimulated vascular endothelial cell injury by inhibiting proliferation and function [44]. Whether the fetoplacental endothelial dysfunction seen in GDM is relevant to the reduced expression of miR-140-3p and miR-574-3p has not been elucidated. We first demonstrated that placenta-derived exosomal miR-140-3p and miR-574-3p suppress the proliferation, migration, and tube information of umbilical vein endothelial cells. Despite reports that miR-140 decreases VEGF expression [44][45][46], the underlying mechanism remains unclear. Similarly, we observed that miR-140-3p decreased VEGF expression in endothelial cells. miR-140-3p regulates the expression of ZO-1-associated kinase (ZAK) and Forkhead box protein Q1 (FOXQ1) by targeting their mRNA 3 UTR [41,47]. In this investigation, we found that miR-140-3p binds to the 3 UTR of VEGF mRNA, which suggests that miR-140-3p reduces VEGF expression by binding directly to VEGF mRNA. MiR-574-3p regulates VEGFA translation by competitively binding hnRNP L [33]. Moreover, our results show that miR-574-3p downregulates VEGF expression by targeting hnRNP L. miR-140-3p and miR-574-3p combined treatment inhibited VEGF expression in umbilical vein endothelial cells more significantly than miR-140-3p or miR-574-3p alone; thus, the reduced levels of miR-140-3p and miR-574-3p offset the inhibition of VEGF expression, resulting in placental-derived exosome-mediated fetoplacental endothelial dysfunction in GDM.
Chemerin, a newly discovered adipocytokine, is involved in metabolic diseases and inflammation regulation. It promotes the chemotaxis of macrophages and immature dendritic cells in a chemR23-dependent manner [48]. Studies have shown that chemerin expression is increased in GDM [27,49]. We previously found that chemerin reduced the number of neurons by inducing macrophage recruitment, activation of pyroptosis, and the release of inflammatory cytokines in the fetal brain tissue of offspring of diabetic dams, in turn leading to cognitive impairment [20]. However, it is not clear whether chemerin is associated with placental inflammation in GDM. Additionally, the NRLP3 inflammasome may participate in this process, the assembly of which results in the caspase 1-dependent release of IL-1β and IL-18 [50]. This study confirmed that chemerin recruited macrophages and promoted NLRP3 expression, caspase 1 activation, and the release of inflammatory factors IL-1β and IL-18 in the placental tissue of GDM, thereby inducing placental inflammation. Few studies have shown the interaction between chemerin and miRNAs, and one has reported that chemerin decreased miR-217 expression and promoted proliferation, adipogenesis, and angiogenesis in 3T3-L1 preadipocytes [51]. Several groups have reported that exosomal miRNAs are regulated by proinflammatory cytokines [52,53]. We found that trophoblast cell-derived exosomal miR-140-3p and miR-574-3p were strongly downregulated by the supernatant from macrophages treated with chemerin plus IL-18 and/or IL-1β stimulation. Meanwhile, the levels of placenta-derived exosomal miR-140-3p and miR-574-3p were negatively related to chemerin from clinical samples. This suggests that chemerin inhibits trophoblast cell-derived exosomal miR-140-3p and miR-574-3p expression by inducing placental inflammation.
This study has several limitations, including the relatively low number of subjects, inclusion of subjects with a relatively high maternal age, and smaller gestational weight gain of the GDM group relative to the control. Second, chemerin-induced placental macrophage recruitment was explored only in the placenta tissue of pregnant women by immunofluorescence; ideally, these results should be verified in animal experiments. Thus, additional studies are needed to reveal the specific mechanism underlying chemerin-induced placental inflammation. Third, we found that IL-1β and IL-18 reduced the expression of exosomal miR-140-3p, miR-574-3p, and pri-miRNA processing-related proteins (including Drosha, DGCR8, and Exportin-5) in trophoblast cells, which suggests that IL-1β and IL-18 alter trophoblast cell-derived exosomal miR-140-3p and miR-574-3p expression by regulating trophoblast pri-miR-140-3p and pri-miR-574-3p processing and nuclear export. However, more experiments are needed to confirm this. In addition, IL-1β and IL-18 had no effect on the expression levels of pri-miR-140-3p and pri-miR-574-3p; whether they are related to the reduced transcriptional activity of miR-140-3p and miR-574-3p or accumulation of nuclear pri-miR-140-3p and pri-miR-574-3p induced by processing blockade requires further study.
In conclusion, chemerin reduced placenta-derived exosomal miR-140-3p and miR-574-3p levels by inducing placental recruitment of macrophages and the release of inflammatory factors, thereby increasing VEGF expression and promoting the proliferation, migration, and tube formation of umbilical vein endothelial cells in the GDM group. Our results indicate that placenta-derived exosomal miR-140-3p and miR-574-3p have potential as markers of GDM and that their agomirs could reduce the incidence of offspring complications by attenuating the function of umbilical vein endothelial cells.
Author Contributions: All authors have contributed to this manuscript; L.Z., D.C. and Z.L. conceived the idea and designed the study. L.Z. and Q.W. performed the majority of experiments, performed statistical analysis and prepared figures and tables. S.Z., Y.T. and Y.C. performed miRNA profiling and Western blotting experiments. L.Z. wrote the manuscript. The final version of the manuscript was approved by all authors. Z.L. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. All authors have read and agreed to the published version of the manuscript.