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.

Keywords: Preeclampsia; lncRNA; miR-485-5p/aim2 axis; neat1; treg/th17.

Figure 1.

NEAT1 expression is upregulated in PE and associated with Treg/Th17 balance. (a) heatmap analysis showing lncRNA expression profile in patients with PE and healthy pregnant women. (b) the levels of NEAT1 were examined using RT-qPCR in CD4⁺ T cells isolated from patients with PE and healthy pregnant women. (c) Treg cells were assessed by flow cytometry and quantified. (d) correlation analysis evaluated the interaction between NEAT1 and Treg cells. (e) Th17 cells were analyzed using flow cytometry and quantified. (f) Correlation analysis between NEAT1 and Th17 cells. (g) calculation of the ratio of Treg/Th17. (h) correlation analysis evaluated the relationship between NEAT1 and Treg/Th17 ratio. ***P < 0.001

Figure 2.

Knockdown of NEAT1 promotes the balance of Treg/Th17. (a) transfection efficiency was determined using RT-qPCR. (b) Treg and Th17 cells were assessed by flow cytometry. the percentage of (c) Treg cells and (d) Th17 cells was quantified. (e) the ratio of Treg/Th17 was calculated. ***P < 0.001. **P < 0.01

Figure 3.

NEAT1 acts as a miR-485-5p sponge. the interaction between NEAT1 and miR-485-5p was (a) predicted using the starbase dataset and (b) confirmed using dual-luciferase reporter analysis. (c) miR-485-5p levels were detected by RT-qPCR with knockdown of NEAT1. (d) miR-485-5p was measured in CD4⁺ T cells isolated from patients with PE and healthy pregnant women. (e) Correlation analysis between NEAT1 and miR-485-5p in PE. ***P < 0.001. **P < 0.01

Figure 4.

Knockdown of NEAT1 improves Treg/Th17 imbalance via sponging of miR-485-5p. (a) transfection efficiency was examined by RT-qPCR in CD4⁺ T cells following transfection with inhibitor NC or miR-485-5p inhibitor. (b) Treg and Th17 cells were assessed using flow cytometry. (c) quantification of the percentage of Treg cells. (d) quantification of the percentage of Treg cells. (e) calculation of the ratio of Treg/Th17. **P < 0.01 vs. inhibitor NC group in (A). ***P < 0.001, **P < 0.01 and *P < 0.05 vs. si-NC + inhibitor NC group. ###P < 0.01 and ##P < 0.01 vs. si-NEAT1 + inhibitor NC group

Figure 5.

AIM2 is a miR-485-5p target. (a) the predicted binding sites of AIM2 in miR-485-5p were analyzed using the targetscan dataset. (d) the luciferase activity was assessed when co-transfected with WT-AIM2 or MUT-AIM2 and mimics or NC. (c) AIM2 was tested by RT-qPCR when inhibiting miR-485-5p. (D) AIM2 protein expression was detected using western blot when inhibiting miR-485-5p. (e) AIM2 protein levels were measured by western blot when knockdown of NEAT1. (f) AIM2 was measured in CD4⁺ T cells isolated from patients with PE and healthy pregnant women. (g) Correlation analysis between AIM2 and miR-485-5p expression in PE. ***P < 0.001. **P < 0.01

Figure 6.

Knockdown of NEAT1 improves Treg/Th17 imbalance through modulation of AIM2. (a) Transfection efficiency was examined by RT-qPCR in CD4⁺ T cells following transfection of pcDNA3.1 or pcDNA3.1-AIM2. (b) Treg and Th17 cells were assessed using flow cytometry. the percentage of (c) Treg cells and (d) Th17 cells was quantified. (e) calculation of the ratio of Treg/Th17. ***P < 0.001 vs. pcDNA3.1 group in (A). ***P < 0.001, **P < 0.01 and *P < 0.05 vs. si-NC + pcDNA3.1 group. ###P < 0.01 and ##P < 0.01 vs. si-NEAT1 + pcDNA3.1 group