Silencing of long non-coding RNA NEAT1 improves Treg/Th17 imbalance in preeclampsia via the miR-485-5p/AIM2 axis

ABSTRACT T-regulatory (Treg)/T-helper 17 (Th17) imbalance is associated with preeclampsia (PE). Herein, we aimed to explore the effect and mechanism of lncRNA NEAT1 on the Treg/Th17 balance. The levels of nuclear enriched abundant transcript 1 (NEAT1), miR-485-5p, and absent in melanoma 2 (AIM2) in CD4+ T cells were determined using real-time quantitative polymerase chain reaction (RT-qPCR). Treg and Th17 cells were examined using flow cytometry. The relationship between miR-485-5p and NEAT1 or AIM2 was assessed using a dual-luciferase reporter assay. Pearson’s correlation coefficient was used to analyze the correlation. All the data indicated that NEAT1 was upregulated in PE. The number of Treg cells decreased and was negatively related to NEAT1, whereas the number of Th17 cells increased and was positively related to NEAT1 in PE. Knockdown of NEAT1 increased the Treg cells and Treg/Th17 but decreased Th17 cells. Furthermore, NEAT1 sponges miR-485-5p to suppress the target AIM2 levels. Inhibition of miR-485-5p or upregulation of AIM2 abrogated the effect on Treg/Th17 balance induced by knockdown of NEAT1. In conclusion, silencing of NEAT1 promoted Treg/Th17 balance via the miR-485-5p/AIM2 axis in PE, suggesting that NEAT1 is a potential target for the treatment of PE.


Introduction
Preeclampsia (PE) is a common complication of pregnancy that manifests as multiple organ dysfunction [1]. It is defined as hypertension after 20 weeks of gestation with proteinuria of no less than 0.3 g/day [2]. PE can be divided into early or late onset, diagnosed before or after 34 weeks of gestation, respectively [3]. Globally, PE affects about 4% of pregnant women every year and may lead to perinatal maternal and fetal death [4]. Without timely, effective prevention and treatment, PE develops into more severe eclampsia with seizures [5]. However, because the pathogenesis of PE remains unclear, there is currently no complete cure for PE. The only clinical solution is to end the pregnancy and deliver the fetus and placenta [1]. Therefore, it is necessary to further explore the pathogenesis of PE and identify effective treatment strategies.
Long non-coding RNAs (lncRNAs) are transcripts with lengths of > 200 nt. They can only modulate gene expression in the form of RNA, and do not encode proteins. Recent studies have shown the involvement of lncRNAs in epigenetic regulation, chromatin modification, and transcriptional activation or interference [6]. Dysregulation of lncRNAs is implicated in the incidence and progression of diseases, such as cardiovascular diseases, degenerative diseases, diabetes, and cancer [7,8]. LncRNAs play significant roles in PE by regulating cellular processes, such as proliferation, differentiation, metastasis, and apoptosis [9,10]. The lncRNA NEAT1 is a structural part of paraspeckles and is crucial for RNA stability, isoform conversion, and paraspeckle assembly [11]. NEAT1 participates in the pathogenesis of malignancies [12]. However, the specific function of NEAT1 in PE has not yet been elucidated.
T-helper 17 (Th17) and T-regulatory (Treg) cells differentiate from T cells. Th17 cells promote inflammation through the production of IL-17, whereas Treg cells are specific suppressors of inflammation [13]. Balance between Tregs and Th17 cells is involved in immune homeostasis [14]. The differentiation of Treg and Th17 cells depends on the upregulation of Foxp3 in Tregs and RORγt in Th17 cells [15]. Treg/Th17 imbalance is associated with PE, which may be due to improper activation of the immune system during PE onset [16]. However, the mechanism of Treg/ Th17 in PE remains unclear.
In this study, we aimed to explore the relationship between NEAT1 and Treg/Th17 balance in PE. We assumed that silencing of NEAT1 prevents Treg/Th17 imbalance in PE and the miR-485-5p/ AIM2 axis is the molecular mechanism. These findings suggest that NEAT1 is a potential target for the treatment of PE.

Subjects
This study was approved by the Shenzhen Longhua District Central Hospital (approval code No. 2,018,102,503). Written informed consent was obtained from each subject prior to the study. Participants included pregnant women (n = 25) diagnosed with PE, as well as 25 healthy pregnant women as a control. Subjects enrolled in our study were natural singleton pregnancy. Patients with the following diseases were excluded from this study: autoimmune diseases, pre-pregnancy chronic hypertension, cancer, and other pregnancy complications. Whole blood samples were collected from all subjects.

Cell collection and culture
Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy pregnant women and patients with PE. The isolation was performed using Ficoll-Hypaque with density gradient centrifugation. Venous blood (2 mL) collected from all subjects was mixed with Hank's solution (2 mL). After adding 2 mL Ficoll-Hypaque solution (MP Biomedicals, Aurora, OH, USA), the samples were centrifuged at 2000 r/min for 5 min. Next, the mononuclear cell layer was transferred to another centrifuge tube, mixed with Hank's solution, and centrifuged at 2000 r/min for 5 min. After washing with Hank's solution, PBMCs were maintained in PBMC complete medium (Procell, Wuhan, China) at 37°C with 5% CO 2 .
CD4 + T cells were isolated from PBMCs using the EasySep™ Human CD4 + T Cell Isolation Kit (StemCell Technologies, Vancouver, BC, Canada). Briefly, 5 х 10 7 cells were incubated with cocktail (50 μL) for 5 min and the samples were mixed with RapidSpheres (50 μL) and the EasySep™ Buffer was added up to 2.5 mL. The samples were then incubated with a magnet for 3 min at 25°C. After removing the magnet, an enriched cell suspension remained.

Dual-luciferase reporter assay
Wild type (WT) and mutant sequences of NEAT1 and AIM2 were cloned into the Pmir-Glo vector (Promega, Madison, USA) to construct the recombinant plasmids. HEK293T cells were cotransfected with mimics or NC and WT or mutant (MUT) reporter plasmids using Lipofectamine 2000 (Invitrogen, Carlsbad, USA) for 48 h. The fluorescence signals were measured using the Luciferase Assay System (Promega).

Western blot
Total protein was isolated using RIPA lysis buffer (Beyotime, Shanghai, China). After testing the protein concentration using BCA Kit (TIANGEN), each protein sample was separated using 10% SDS-PAGE and transferred onto PVDF membranes. After blocking using 5% nonfat milk, the membranes were incubated with anti-AIM2 at 4°C overnight and then incubated with secondary antibody at 25°C for 2 h. Protein bands were visualized using ECL reagent (Beyotime) and quantified using Image J software (version 1.8.0; National Institutes of Health, Bethesda, Maryland, USA).

Statistical analysis
Data were analyzed using GraphPad Prism 8.0 software (San Diego, USA) and are shown as mean ± SD. Student's t-test and one-way ANOVA were used to evaluate the significance between and among groups, respectively. Pearson's correlation coefficient was used to analyze the correlation.

Results
The present study aimed to investigate the the effect of NEAT1 on Treg/Th17 balance in PE and further revealed the potential molecular mechanism. In this study, we found a loss of NEAT1 improves Treg/Th17 imbalance by regulating the miR-485-5p/AIM2 axis in PE. This study may provide a new insight to treat PE.

NEAT1 is upregulated in PE and associated with Treg/Th17 balance
According to the microarray results, NEAT1 expression was predicted to be higher in patients with PE than in healthy pregnant women (Figure 1(a)). The results of RT-qPCR proved that NEAT1 expression was significantly elevated in the PE group (P < 0.001; Figure 1(b)). The percentage of Treg cells significantly decreased in PE (P < 0.001), which negatively correlated with NEAT1 levels (P = 0.0003, R = −0.6650; Figure 1(c-d)). In contrast, the percentage of Th17 cells significantly increased in PE (P < 0.001), which positively correlated with NEAT1 levels (P = 0.0008, R = 0.6246; Figure 1(e-f)). The ratio of Treg/Th17 was significantly reduced in patients with PE (P < 0.001), which negatively correlated to NEAT1 levels (P < 0.0001, R = −0.7067; Figure 1(g-h)).

Knockdown of NEAT1 improves Treg/Th17 imbalance through modulation of AIM2 expression
AIM2 levels were significantly higher in the pcDNA3.1-AIM2 group than in the pcDNA3.1 one (P < 0.001; Figure 6(a)). The results of flow cytometry showed that overexpression of AIM2 abolished the increased number of Treg cells and decreased number of Th17 cells induced by NEAT1 knockdown (Treg: P < 0.001; Th17: P < 0.01; Figure 6(b-d)). The Treg/Th17 balance was markedly promoted by knockdown of NEAT1, while overexpression of AIM2 reversed this effect (P < 0.01; Figure 6(e)).

Discussion
In this study, we first observed that NEAT1 silencing promoted the balance of Treg/Th17 cells in PE. Furthermore, the miR-485-5p/AIM2 axis was first identified as the underlying mechanism.
Abnormal activation of the maternal immune system causes placental dysfunction, leading to the pathogenesis of PE, followed by a systemic inflammatory response [3,18]. Thus, Treg/Th17 imbalance is a mechanism of PE, manifesting as impaired Treg activity and excessive Th17 response [19]. Studies have reported that inducing an imbalance in Treg/Th17 using low vitamin D, CD81, and alteration of PD-1/PD-L1 contributes to PE [20][21][22]. In this study, we found that the percentage of Treg cells decreased, but that of Th17 increased, proving that Treg/Th17 imbalance is involved in PE.
NEAT1 is a newly identified immune regulator that affects mononuclear-macrophage function as well as T cell differentiation [23]. During the immune response, NEAT1 can promote the activation of inflammasomes, such as NLRP3 and NLRC4 in macrophages [24]. Additionally, NEAT1 inhibits the inflammatory response by regulating macrophage polarization [25]. A previous study revealed that knockdown of NEAT1 inhibits Th17/CD4 + T cell differentiation [26]. However, the effects of NEAT1 on Treg/Th17 balance remain unknown. NEAT1 is upregulated in the placenta of rats with PE and inhibits the growth of trophoblast cells [27]. Herein, we found that NEAT1 was increased in patients with PE, consistent with results found in the rat placenta. Moreover, we found that knockdown of NEAT1 improved Treg/Th17 imbalance, providing a new insight into NEAT1 function in PE.
AIM2, an innate immune sensor, can identify DNA from microbes and hosts, and is arguably a specific marker of innate immune initiation [34,35]. The activation of AIM2 causes the secretion of pro-inflammatory cytokines and induces cell pyroptosis [36]. The AIM inflammasome responds to various diseases, such as skin disease, diabetes, cancer, and infectious diseases [37]. However, the involvement of AIM2 in Treg/Th17 balance remains unknown. In the current study, AIM2 was found to be a target of miR-485-5p. The level of AIM2 was increased in PE, consistent with a previous study [38]. Furthermore, overexpression of AIM2 abolished the effects on Treg/Th17 balance induced by knockdown of NEAT1. Taken together, these findings suggest that silencing of NEAT1 improves Treg/Th17 imbalance by sponging miR-485-5p.

Conclusion
NEAT1 expression was upregulated in PE and associated with a Treg/Th17 imbalance. Furthermore, silencing of NEAT1 promoted the balance of Treg/Th17 cells by modulating the miR-485-5p/AIM2 axis. These findings suggest that NEAT1 is a potential target for the treatment of PE.