Lncrna NEAT1 Regulates Th1/Th2 in Pediatric Asthma by Targeting MicroRNA-217/GATA3

Abstract

Background: The imbalance of immune response between helper Th1 and Th2 cells is the direct cause of asthma. It was closely related to abnormal expression of lncRNAs. However, whether lncRNAs can regulate Th1/Th2 balance in pediatric asthma remains to be investigated.

Methods: Peripheral blood samples were collected from children with asthma and normal volunteers at the Children's Hospital of Shaanxi Provincial People's Hospital (Xi'an, China) in 2020. The qRT-PCR was used to detect the expression of lncRNA NEAT1, miR-217 and GATA3 in peripheral blood samples. The effects of lncRNA NEAT1, miR-217, and GATA3 on CD4+T cell population were detected in vitro. Meanwhile, the regulatory effect of lncRNA NEAT1/miR-217/GATA3 was evaluated through the dual luciferase report assay, functional assays and animal experiments.

Results: We investigated that lncRNA NEAT1 and GATA3 was significantly up-regulated in CD4+T cells in peripheral blood of children with asthma (P<0.001). Knockdown of lncRNA NEAT1 or GATA3 significantly reduced Th2-related cytokines (P<0.05), but had no effect on Th1 cells. Importantly, the interactions of lncRNA NEAT1 with miR-217 and miR-217 with GATA3 were confirmed by dual luciferase report assay. Meanwhile, functional assays and animal experiments demonstrated that lncRNA NEAT1 regulated GATA3 expression through sponge miR-217, thereby regulating Th1/Th2 balance in CD4+T cells in pediatric asthma.

Conclusion: lncRNA NEAT1/miR-217/GATA3 axis may reveal the immunological mechanism of pediatric asthma, which has potential clinical application value in the future.

Keywords: Immunology; Long noncoding RNAs; Pediatric asthma; Proteins.

Fig. 1:

Effect of downregulation of lncRNA NEAT1 on Th1/Th2 balance in pediatric asthma. (A) qRT-PCR detected lncRNA LAMAT1, lncRNA NEAT1, lncRNA-GAS5 and lncRNA XIST expression in CD4+T cells from 10 pairs of peripheral blood samples from pediatric asthma patients and normal children. (B) NEAT1 expression in transfected CD4+T cells were examined using qRT-PCR. (C-E) Th2-related cytokines (IL-4 and IL-10) and Th1-associated cytokine (INF-γ) were detected using ELISA assay. (F-G) The percentage of Th1 and Th2 cell were measured by flow cytometry. Data are shown as mean ± SD (n=3). **P<0.01; ***P<0.001

Fig. 2:

LncRNA NEAT1 negatively regulates expression of miR-217. (A) qRT-qPCR detected miR-217 expression in CD4+T cells from pediatric asthma patients and normal children. (B) miR-217 expression in CD4+ T cells transfected with miR-217 mimic were examined using qRT-PCR. (C–E) Th1/2-related cytokines were detected by ELISA assay. (F–G) Flow cytometry was used to detect the effect of miR-217 mimic on the percentage of Th1 and Th2 cells. (H) The binding site between NEAT1 and miR-217 was predicted by RNAhybrid database. (I) Dual luciferase reporter assay validated the target relationship between NEAT1 and miR-217. (J) The qRT-qPCR analysis of miR-217 expression in CD4+T cells transfected with siRNA-NEAT1. (K) qRT-qPCR analysis of NEAT expression in CD4+T cells transfected with miR-217 mimic. Data are shown as mean ± SD (n=3). **P<0.01; ***P<0.001

Fig. 3:

miR-217 affects Th1/Th2 cells by regulating GATA3 expression. (A) qRT-qPCR detected GATA3 expression in CD4+T cells from pediatric asthma patients and normal children. (B) GATA3 expression were examined using western blot after CD4+T cells transfected with si-GATA3-1/2. (C–E) Th1/2-related cytokines were detected by ELISA assay. (F–G) Flow cytometry was used to detect the effect of GATA3 knockdown on Th1 and Th2 cell percentages. (H) The binding site between miR-217 and GATA3 was predicted by RNAhyrid website. (I) The Dual luciferase reporter assay to validate target relationship between miR-217 and GATA3. (J) The RT-qPCR analysis of miR-217 expression in CD4+T cells transfected with siRNA-NEAT1 and siRNA-NEAT1+miR-217 inhibitor. Data are shown as mean ± SD (n=3). **P<0.01; ***P<0.001

Fig. 4:

lncRNA NEAT1 and miR-217 competitively regulated GATA3 CD4+T cells were transfected with Si-NC, Si-NEAT1, Si-NEAT1+miR-217 inhibitor, and Si-NEAT1+miR-217 inhibitor+Si-GATA3, respectively. (A–C) ELISA assay of Th1/2-associated cytokine. (D–E) The percentage of Th1 and Th2 cells in CD4+T cells using flow cytometry. Data are shown as mean ± SD (n=3). **P<0.01; ***P<0.001

Fig. 5:

lncRNA NEAT1 regulates Th1/Th2 balance in pediatric asthma by targeting miR-217/GATA3 in vivo. A mouse asthma model was constructed by OVA, followed by intraperitoneal injection of si-NEAT1, miR-217 inhibitor or si-GATA3, respectively. At the end of experiment, the peripheral blood of mice was collected to isolate CD4+T cells. (A–C) Th1/2-related cytokines were detected by ELISA. (D–E) The percentage of Th1 and Th2 cells detected using flow cytometry. (F) H&E staining of mouse lung tissues in each group (magnification: 200X). Data are shown as mean ± SD (n=3). *P<0.05; **P<0.01; ***P<0.001