The regulatory network among CircHIPK3, LncGAS5, and miR-495 promotes Th2 differentiation in allergic rhinitis

Abstract

Allergic rhinitis (AR) is a common allergic disease which is characterized by the promotion of Th2 differentiation of CD4⁺ T cells. However, the mechanisms underlying Th2 differentiation remain unclear. Non-coding RNAs play a critical role in Th2 differentiation, whereas few studies have revealed the interactions among long non-coding RNAs, circular RNAs, and microRNAs. In this study, the differential expressions of several circRNAs and lncRNAs were compared in nasal mucosa samples of AR patients and mice with experimentally induced AR as compared to healthy controls. The results showed that the highly expressed CircHIPK3 and LncGAS5 promoted Th2 differentiation of ovalbumin-induced CD4⁺ T cells and aggravated nasal symptoms of AR mice. We also found that CircHIPK3 and LncGAS5 induced the upregulation of Th2 cell-specific transcript factor GATA-3 via modulating their common target miR-495. Meanwhile, the intranasal administration of CircHIPK3 or LncGAS5 knockdown lentivirus decreased nasal symptoms of AR mice. In conclusion, our findings indicated that the interactions among CircHIPK3, LncGAS5, and miR-495 play a critical role in the regulation of Th2 differentiation in AR.

Fig. 1. Identification of circRNAs and lncRNAs differentially expressed in AR nasal mucosa.

Clinical samples of nasal mucosa were scraped from the surface of the inferior nasal turbinate of AR patients (n = 10) and healthy individuals (n = 10) using a plastic curette. Mouse AR was induced by OVA sensitization and OVA intranasal challenge. The murine samples of nasal mucosa were collected from AR-induced mice (n = 6) and control mice (n = 6). a, b The expressions of five circRNAs and five lncRNAs in mucosa samples were detected using qRT-PCR. c The expression of IL-4 and GATA-3 in mucosa samples was detected using qRT-PCR and western blot analysis, respectively. *p < 0.05, **p < 0.01 vs Control.

Fig. 2. Knockdown of CircHIPK3 or LncGAS5 alleviates nasal symptoms of AR mice.

AR mice were intranasally treated with CircHIPK3 knockdown lentivirus (lenti-siCircHIPK3) or LncGAS5-knockdown lentivirus (lenti-siLncGAS5) (n = 6 in each group). a, b The number of sneezes and nasal rubbings. c H&E staining of the nasal mucosa (Scale bar = 10 μm). d, e Levels of IgE and IL-4 in serum and nasal mucosa were detected using ELISA. **p < 0.01 vs Control + lenti-GFP. ^##p < 0.01 vs AR + lenti-GFP.

Fig. 3. Overexpression of CircHIPK3 or LncGAS5 promotes Th2 differentiation of OVA-induced CD4⁺ T cells.

CD4⁺ T cells, isolated from PBMCs of AR patients or spleen tissues of mice, were induced by OVA and Th2 inducing agents for 7 days after the transfection of CircHIPK3 lentivirus (lenti-CircHIPK3) or LncGAS5 lentivirus (lenti-LncGAS5). a, b The percentage of Th2 cells, the protein level of GATA-3, and the supernatant level of IL-4 were detected using flow cytometry, western blot analysis, and ELISA, respectively. **p < 0.01 vs OVA + lenti-GFP.

Fig. 4. CircHIPK3 and LncGAS5 interact directly with miR-495 and regulate its expression.

a The predicted binding sequences and the artificially mutated sequences between CircHIPK3/LncGAS5 and miR-495. b The relative luciferase activity after the co-transfection with miR-495 mimic and the CircHIPK3/LncGAS5 mutant/wild type (MUT/WT) reporter vector. c The relative miR-495 level in the complex which was pulled down by CircHIPK3 or LncGAS5 probe using the RAP assay. d Relative expression of CircHIPK3 and LncGAS5 in the complex which was pulled down by biotin-labeled miR-495 using the RNA pull-down assay. e The cellular distribution of CircHIPK3, LncGAS5, and miR-495 in OVA and Th2 differentiation-induced CD4⁺ T cells using the FISH assay. f Relative expression of LncGAS5 and miR-495 after CircHIPK3 knockdown or overexpression. g Relative expression of CircHIPK3 and miR-495 after LncGAS5 knockdown or overexpression. **p < 0.01 vs negative controls (pre-NC, Biotin NC, or NC). ^##p < 0.01 vs lentivirus control (Lenti-GFP).

Fig. 5. CircHIPK3 and LncGAS5 increase GATA-3 expression via downregulating miR-495.

a The predicted binding sequences and the artificially mutated sequences between miR-495 and the 3′-untranslated region (UTR) of GATA-3. b The relative luciferase activity after the co-transfection with miR-495 mimic and the GATA-3 3′-UTR mutant/wild type (MUT/WT) reporter vector. c The relative luciferase activity after the co-transfection with miR-495 inhibitor and the GATA-3 3′-UTR mutant/wild type (MUT/WT) reporter vector. d CD4⁺ T cells were transfected with Lenti-CircHIPK3/Lenti-LncGAS5 or co-transfected with miR-495 mimic followed by the induction of OVA and Th2 differentiation. The GATA-3 protein level was detected using western blot analysis. e CD4⁺ T cells were also transfected with si-CircHIPK3/si-LncGAS5 or co-transfected with miR-495 inhibitor followed by the induction of OVA and Th2 differentiation. The GATA-3 protein level was detected using western blot analysis. **p < 0.01 vs negative controls (pre-NC or NC).

Fig. 6. CircHIPK3 and LncGAS5 promote Th2 differentiation of OVA-induced CD4⁺ T cells via miR-495/GATA-3 pathway.

a CD4⁺ T cells were induced by OVA and Th2 inducing agents for 7 days after being transfected with Lenti-CircHIPK3/Lenti-LncGAS5 or co-transfected with miR-495 mimic. The percentage of Th2 cells and the supernatant level of IL-4 were detected using flow cytometry, and ELISA, respectively. b CD4⁺ T cells were induced by OVA and Th2 inducing agents for 7 days after being transfected with si-CircHIPK3/si-LncGAS5 or co-transfected with miR-495 inhibitor. The percentage of Th2 cells and the supernatant level of IL-4 were detected. c CD4⁺ T cells were induced by OVA and Th2 inducing agents for 7 days after being transfected with Lenti-CircHIPK3/Lenti-LncGAS5 or co-transfected with si-GATA-3. The percentage of Th2 cells and the supernatant level of IL-4 were detected. d CD4⁺ T cells were induced by OVA and Th2 inducing agents for 7 days after being transfected with si-CircHIPK3/si-LncGAS5 or co-transfected with Lenti-GATA-3. The percentage of Th2 cells and the supernatant level of IL-4 were detected. **p < 0.01 vs OVA + lenti-GFP + Pre-NC/si-control. ^#p < 0.05 vs OVA + Lenti-CircHIPK3/Lenti-LncGAS5 + Pre-NC or OVA + si-CircHIPK3/si-LncGAS5 + si-control.

Fig. 7. Knockdown of CircHIPK3 or LncGAS5 alleviates nasal symptoms of AR mice via miR-495/GATA-3 pathway.

a AR-induced mice were treated intranasally with CircHIPK3/LncGAS5-knockdown lentivirus, or together with miR-495 inhibitor (n = 6 in each group). The H&E staining of nasal mucosa (Scale bar = 10 μm), the number of sneezes and nasal rubbings, and the levels of IgE and IL-4 in serum and nasal mucosa were shown. b AR-induced mice were treated intranasally with CircHIPK3/LncGAS5-knockdown lentivirus, or together with GATA-3-overexpressing lentivirus (n = 6 in each group). The H&E staining of the nasal mucosa (Scale bar = 10 μm), the number of sneezes and nasal rubbings, and the levels of IgE and IL-4 in serum and nasal mucosa were shown. *p < 0.05, **p < 0.01 vs AR + lenti-GFP + NC or AR + lenti-GFP. ^#p < 0.05 vs AR + Lenti-siCircHIPK3/Lenti-siLncGAS5 + NC or AR + Lenti-siCircHIPK3/Lenti-siLncGAS5.