Long non-coding RNA MALAT1 promotes Th2 differentiation by regulating microRNA-135b-5p/GATA-3 axis in children with allergic rhinitis

Allergic rhinitis (AR) threatens patient survival. CD4 + T cells play key roles in AR progression. Long non-coding RNAs (lncRNAs) are key regulators of cell differentiation. Therefore, we investigated the molecular mechanism of the lncRNA metastasis-associated (MALAT1) in AR. Expression levels of MALAT1, microRNA (miR)-135b-5p, interleukin-4 (IL-4), and GATA-binding protein 3 (GATA-3) in the nasal mucosa of AR patients were quantified. CD4 + T cells were isolated from the peripheral blood of healthy volunteers and treated with ovalbumin (OVA) and Th2 inducers. After MALAT1 and miR-135b-5p levels changed in CD4 + T cells, the proportion of IL-4-expressing cells and the levels of IL-4 and GATA-3 in OVA-induced CD4 + T cells were determined. Binding relationships among MALAT1, miR-135b-5p, and GATA-3 were predicted and verified. Rescue experiments were performed to confirm the role of the MALAT1/miR-135b-5p/GATA-3 axis in Th2 differentiation of CD4 + T cells. MALAT1, IL-4, and GATA-3 expression was upregulated, whereas miR-135b-5p expression was downregulated, in patients with AR. MALAT1 knockdown or miR-135b-5p overexpression in CD4 + T cells notably decreased the proportion of IL-4-expressing cells and downregulated GATA-3 and IL-4 expression in OVA-induced CD4 + T cells. MALAT1 and GATA-3 exhibited competitive binding toward miR-135b-5p. MALAT1 facilitated CD4 + T cell Th2 differentiation via the miR-135b-5p/GATA-3 axis. MALAT1 facilitated AR development by facilitating CD4 + T cell Th2 differentiation via the miR-135b-5p/GATA-3 axis. This study may provide guidance for clinical treatment of AR.


| INTRODUCTION
Allergic rhinitis (AR), the most prevalent chronic disease in childhood, notably reduces the quality of life of children. 1,2 Children with AR may experience sleep disorders, and the severity of AR is correlated with the intensity of sleep disorders. 3 AR is closely associated with asthma, with asthma representing the main risk factor for AR onset. 2 The main symptoms of AR include nasal congestion, rhinorrhea, and sneezing. 4 AR is defined as an immunoglobulin E (IgE)-mediated inflammatory disease of the nasal wall and is caused by the inhalation of allergens in the environment. 5,6 Dust mites, pollen, molds, and animals may trigger AR symptoms. 7 Furthermore, early exposure to antibiotics within the first year of life contributes to AR development in children. 8 Nevertheless, AR treatment primarily focuses on the use of desensitizing drugs, which only target the symptom. 9 Therefore, it is essential to explore the underlying mechanism of AR to optimize AR treatment in children.
CD4 + T cells, which are crucial components of the immune system, play a role in inflammatory disorders. 10 Several studies have shown that CD4 + T cells play a crucial role in AR. 11,12 Specifically, CD4 + T cells can differentiate into several helper subsets (T-helper [Th]1, Th2, Th17, regulatory T, and T follicular helper cells) upon activation by pathogens in the microenvironment. 13,14 AR is characterized by an imbalance of Th1/Th2 differentiation, with a predominance of Th2 differentiation. 15 Therefore, this study was designed to explore the molecular mechanism of CD4 + T cell Th2 differentiation in AR with the aim of developing novel approaches for more effective treatment of AR in children.
Long non-coding RNAs (lncRNAs) are potent regulators of cell differentiation, response to stimulation, and immune response. 16 As shown previously, lncRNAs play key roles in AR pathogenesis. 17 The lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been reported to play critical roles in multiple physiological processes. 18 MALAT1 is also involved in various inflammatory diseases. 19,20 MALAT1 regulates the Th1/Th2 balance within CD4+ T cells by decoying miR-155. 21 Th2 differentiation involves transcription factors such as GATA-binding protein 3 (GATA-3), and GATA-3 can increase the secretion of interleukin-4 (IL-4) by Th2 cells. 22 Additionally, the role of microRNAs (miRNAs) in modulating allergic inflammation in AR is gaining attention. 23,24 miR-135b has been shown to be involved in asthma. 25 Nevertheless, the interplay between MALAT1 and miR-135b-5p in AR remains unclear.
Therefore, we speculated that MALAT1 might regulate CD4 + T cell Th2 differentiation in AR, depending on miR-135b-5p-mediated GATA-3. Consequently, we performed a series of histological and molecular experiments to identify the underlying molecular mechanisms of the involvement of MALAT1 in Th2 differentiation in AR to develop novel therapies against AR progression.

| Clinical samples
Nasal mucosa tissues were scraped from the inferior turbinate surface of 10 children with AR and 10 healthy children without AR using a plastic curette. The diagnostic criteria for AR were as follows: (1) a typical AR history of more than 2 years, (2) a clinical manifestation of runny nose, (3) a positive skin prick test for a specific antigen, and (4) a serum-specific IgE level of more than 0.3 IU/ml. Information on the subjects is presented in Table 1.

| CD4 + T cell isolation and induction for Th2 differentiation
Peripheral blood was collected from healthy volunteers, and peripheral blood mononuclear cells (PBMCs) were obtained by density gradient centrifugation. 26 CD4 + T cells were isolated from PBMCs by magnetic-activated cell sorting (MACS CD4 + T cell Isolation Kit, Miltenyi Biotech), after which CD4 + T cells were cultured for 7 days in RPMI 1640 supplemented with anti-CD3 (3 μg/ml), anti-CD28 (5 μg/ml), IL-4 (20 ng/ml), and anti-IFN-γ (20 μg/ml) to induce Th2 differentiation. Ovalbumin (OVA) (1 μg/ml) was added to induce an inflammatory response in the CD4 + T cells. The culture medium was refreshed every 2-3 days, and the concentrations of the additives were identical in the media. All CD4 + T cells were obtained from healthy volunteers.

| Cell culture
Isolated CD4 + T cells were cultured in RPMI 1640 medium (Sigma-Aldrich). The medium was supplemented with antibiotics (penicillin and streptomycin), glutamine, and fetal bovine serum and replaced every 2-3 days. The cell cultures were incubated at 37 C with 5% CO 2 . The cell concentration was standardized to 1 Â 10 6 /ml before the cells were used for the following experiments.

| Cell transfection
As described in a previous study, 27

| Western blotting
Cells and tissues from each group were lysed using RIPA lysis buffer (Beyotime Biotechnology) and centrifuged to obtain protein samples.
GAPDH was used as the internal reference. The concentration of each protein sample was measured using a bicinchoninic acid kit (Beyotime) to ensure equal amounts of each sample. An appropriate volume of proteins was added and mixed with a loading buffer (Beyotime), which was then heated in a boiling water bath for 3 min to denature the pro-  source=mRNA), the binding site between MALAT1 and miR-135b-5p, as well as that between miR-135b-5p and GATA-3, was predicted.
T A B L E 2 Primer sequence for qRT-PCR Name of primer Sequences Abbreviations: F, forward; qRT-PCR, quantitative reverse transcriptionpolymerase chain reaction; R, reverse.

| Statistical analysis
GraphPad software (version 7.0) was used for data analysis. All data are presented as the mean ± standard deviation (x ± s). T test was utilized for analysis of comparison between two groups. One-way analysis of variance (ANOVA) was used to compare multiple groups, followed by Tukey's multiple comparison test. Statistical significance was set at p < 0.05. 3.2 | MALAT1 and GATA-3 competitively bound to miR-135b-5p Since we found upregulated MALAT1 and GATA-3 in AR tissues, we postulated that MALAT1 might act as a competitive endogenous RNA in AR. The online database starBase (http://starbase.sysu.edu.cn/ F I G U R E 2 MALAT1 competes with GATA-3 to bind to miR-135b-5p. (A) Binding site between miR-135b-5p and MALAT1 3'UTR; (B) binding site between miR-135b-5p and GATA-3 3'UTR; (C) dual-luciferase reporter gene assay was used to confirm the interaction between MALAT1 and miR-135b-5p, **p < 0.01, compared to the OVA + mimic NC + wt-MALAT1 group; (D) RNA pull-down assay was used to verify the interaction between MALAT1 and miR-135b-5p, *p < 0.05, **p < 0.01, compared to the bio-NC-probe group; (E) binding between miR-135b-5p and GATA-3 was verified using dual luciferase reporter gene assay, **p < 0.01, compared to the OVA + mimic NC + wt-GATA-3 group; (F) RNA pull-down assay was used to verify the interaction between miR-135b-5p and GATA-3, *p < 0.05, **p < 0.01, compared to the bio-NC-probe group. CD4 + T cells were transfected with sh-MALAT1 or LV-MALAT1 lentiviral vectors, sh-MALAT1 plus miR-135b-5p inhibitor, or LV-MALAT1 plus miR-135b-5p mimic and then induced with OVA and Th2 inducers.

| DISCUSSION
AR is a common disease that occurs frequently in children and adolescents. 7 CD4 + T cell Th2 differentiation is intrinsically associated with AR occurrence. 15 In the present study, we found that lncRNA MALAT1 promoted CD4 + T cell Th2 differentiation to facilitate AR development by regulating the miR-135b-5p/GATA-3 axis ( Figure 5).
Extensive evidence has validated that MALAT1 is closely associated with inflammation-associated diseases. 28,29 miR-135b-5p has been found to be poorly expressed in different diseases. 30,31 The results of this study revealed that MALAT1 expression was notably elevated and miR-135b-5p expression decreased in patients with AR. In support of these results, MALAT1 has been shown to exhibit a close relationship with chronic rhinosinusitis. 32 MALAT1 is overexpressed and miR-135b is sparsely expressed in asthmatic children. 21,25 Among the subsets of CD4 + T cells, Th2 cells have shown a prominent effect on allergy-associated inflammatory pathologies, such as AR. 12 Differentiation into Th2 cells is intrinsically related to transcription factors, such as GATA-3 (which promotes Th2 cells to secrete IL-4). 22 IL-4 upregulation was previously considered to be positively correlated with AR severity. 33 overexpression reduces IL-4 levels, which is promising for alleviating airway inflammation in asthma. 25 The above data demonstrated that MALAT1 knockdown or miR-135b-5p overexpression inhibited OVAinduced Th2 differentiation of CD4 + T cells.
Subsequently, the underlying mechanism by which MALAT1 and miR-135b-5p influence CD4 + T cell Th2 differentiation in AR was explored. It is well established that lncRNAs can interact with miRNAs to affect the immune system. 16 Moreover, starBase predicted a binding site between MALAT1 and miR-135b-5p. Following a series of experiments, it was identified that MALAT1 targeted miR-135b-5p and that miR-135b-5p overexpression effectively weakened the effect of MALAT1 on IL-4 levels in CD4 + T cells. As shown previously, hsa-miR-135a-5p has a negative correlation with lncRNA MALAT1 in hypertension. 37 However, the interactions between MALAT1 and miR-135b-5p in AR need to be elucidated, which demonstrates the novelty of the present study. However, mounting evidence shows that the transcription factor GATA-3 plays a key role in maintaining Th2 cell identity, exhibiting great significance in AR pathogenesis. 38,39 Therefore, a relationship between GATA-3 and miR-135b-5p was predicted, and GATA-3 was found to be negatively correlated with miR-135b-5p. Consistently, in a previous study, miR-135b was shown to play an inhibitory role on the Th2 master transcription factor GATA-3. 40 Furthermore, suppression of miR-135b-5p rescued GATA-3 downregulation caused by MALAT1 knockdown. GATA-3 knockdown interfered with the effect of MALAT1 overexpression on IL-4 levels in CD4 + T cells. Similarly, it has been reported that MALAT1 sponges a miRNA and raises GATA-3 expression to promote Th2 differentiation in CD4 + T cells during asthmatic inflammation. 21 Therefore, MALAT1 competitively binds to miR-135b-5p and promotes Th2 differentiation of CD4 + T cells by regulating the miR-135b-5p/GATA-3 axis.
Overall, this study suggests that MALAT1 promotes Th2 differentiation within CD4 + T cells by sponging miR-135b-5p to upregulate GATA-3, thereby facilitating AR progression. These results provide a possible explanation for AR development from the perspective of molecular mechanisms and demonstrate that MALAT1 can potentially be used as a predictor for the occurrence of AR. Given the regulatory effects of the MALAT1/miR-135b-5p/GATA-3 axis on CD4 + T cell Th2 differentiation, an important event intrinsically related to AR occurrence, this study revealed novel potential targets for moleculebased therapeutic approaches against AR. Although the findings of the current study provide a new perspective for the treatment of AR, their clinical applicability requires further verification.

ACKNOWLEDGMENTS
Thanks for all the contributors.