Forty-one patients with AR and thirty-nine healthy control subjects were enrolled in the current study. AR was diagnosed according to the Allergic Rhinitis and its Impact on Asthma (ARIA) Guidelines (2016 Revision) after confirmation of disease history, nasal symptoms, clinical examination and skin prick test.4 The healthy control subjects did not have nasal diseases or a history of asthma. Details of the subjects’ characteristics are included in Table 1. Peripheral blood samples collected from the participants were immediately centrifuged to isolate the serum, from which total RNA was extracted for real-time PCR. The study was approved by the ethics committee of our institution, and written informed consent was obtained from each participant (Research Number: 2019-L062).

Thirty 8 week-old BALB/c mice were obtained from the Experimental Animal Center of our university and housed in a specific-pathogen-free biohazard containment facility maintained at a 12 h dark/light cycle and 20° ± 1 °C room temperature with free access to food and water. All animal experiments conducted in this study followed the NIH Guidelines and were approved by the Administration Committee of Experimental Animals of our institution (Research Number: 20180529-001).

The mice were divided into six groups: control, AR (AR mice treated with PBS), mismatched agomir (AR mice treated with mismatched agomir), miR-223-3p agomir (AR mice treated with miR-223-3p agomir), mismatched antagomir (AR mice treated with mismatched antagomir) and miR-223-3p antagomir (AR mice treated with miR-223-3p antagomir). The AR model was induced according to the protocol summarized in Fig. 1.21 In short, mice were sensitized with intraperitoneal injection of 200 μL of saline containing 25 μg ovalbumin (OVA; grade V; Sigma-Aldrich, St. Louis, MO, USA) and 2 mg of aluminum hydroxide gel on days 1, 7 and 14. One week after the primary sensitization, 3% OVA diluted in 20 μL of saline was dripped into the nasal cavities of mice daily from day 21 to day 27 for secondary immunization. For the control (normal) group, only saline was given for intraperitoneal injection and intranasal challenge from beginning to end.

Figure 1.

Schematic diagram of the animal experiment. BALB/c mice were intraperitoneally injected with Ovalbumin (OVA) and aluminum hydroxide (alum) on days 1, 7, and 14 to promote primary sensitization. One week after the final intraperitoneal injection, mice were intranasally challenged with OVA for a week to induce secondary immunization, followed by intranasal treatment using mismatched agomir/miR-223-3p agomir/mismatched antagomir/miR-223-3p antagomir daily within the 3 h of each OVA challenge for 7 days.

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MiR-223-3p agomir, miR-223-3p antagomir, mismatched agomir and mismatched antagomir were synthesized at RiboBio Company (Guangzhou, China). As shown in Fig. 1, mice were treated with an intranasal administration of 5 pmoL/μL miR-223-3p agomir or miR-223-3p antagomir diluted in 20 μL saline within 3 h of OVA challenge daily on days 28‒34. The mismatched agomir/mismatched antagomir groups of mice were intranasally administered 5 pmoL/μL mismatched agomir/mismatched antagomir diluted in 20 μL saline, while the control group was treated with only saline.

Five minutes after the final OVA challenge on day 27, the frequencies of sneezing and nasal rubbing were recorded by a blinded observer twice at 15 min intervals.21,22 Scores were calculated as follows: 1 ‒ Slightly rubs the nose or sneezes less than 3 times; 2 ‒ Rubs the nose repeatedly or sneezes between 3 and 10 times; and 3 ‒ Rubs nose and face seriously or sneezes more than 11 times. Mice with scores greater than 4 were further treated.

Mice were euthanized 24 h after the final intranasal challenge. For RT-PCR, the nasal mucosa of the mice was obtained, immediately immersed in liquid nitrogen and then stored at −80 °C until use. For histological analyses, the heads of the mice were removed and then fixed in 4% paraformaldehyde for 24 h at 4 °C. Nasal tissues of the mice were decalcified, dehydrated in increasing concentrations of ethanol, and then embedded in paraffin.

Total RNA was extracted from cells or the nasal mucosa tissues of the mice with TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and then the concentration and purity of RNA were determined. Serum miRNA was isolated using the miRcute Serum/plasma miRNA isolation kit (TIANGEN Biotech, Beijing, China) according the manufacturer’s instructions. Two micrograms of RNA was reverse transcribed to cDNA, and qRT-PCR was performed according to the instructions for the Power SYBR Green PCR Master Mix (Applied Biosystems, USA). The specific target gene primers are listed in Table 2. The reaction conditions for qRT-PCR were as follows: predenaturation at 95 °C for 10 min, followed by 40 amplification cycles at 95 °C for 20 s, annealing at 62 °C for 30 s and extension at 72 °C for 30 s. For qualification of the expression level of miR-223-3p, the synthetic spike control cel-miR-39 was used as an invariant control for the serum miRNA, while U6 served as the corresponding internal reference for cellular miRNA. The average transcript levels of IL-4, IL-5 and IFN-γ in nasal mucosa and cellular INPP4A were normalized to GAPDH levels. The data were calculated using the 2−ΔΔCT method.

We purchased HEK293T cells from the Cell Bank of Chinese Academy of Medical Science (Shanghai, China) and maintained them in RPMI-1640 medium containing 10% FBS, 100 U/mL penicillin and 100 U/mL streptomycin (Invitrogen) in a humidified incubator with 5% CO2 at 37 °C. In the different groups, transfection was performed using miR-223-3p mimics or miR-223-3p inhibitor or their corresponding negative controls (GeneChem, Shanghai, China) diluted with Opti-MEM I medium (Invitrogen) and transfected into HEK293T cells with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.

To evaluate allergic reactions, serum levels of OVA-specific IgE were measured using solid-phase ELISA kits (CUSABIO, Wuhan, China). Procedures were performed according to the manufacturer’s instructions. Briefly, serum samples were harvested from mice at the time of death and then pipetted into 96 well plates precoated with purified anti-mouse IgE mAb. Anti-mouse IgE-conjugated Horseradish Peroxidase (HRP) was added to the plates. The reactions were developed using 3, 3′, 5, 5′-tetramethylbenzidine and terminated by adding H2SO4. The Optical Density (OD) was recorded by a luminometer set at 450 nm.

For the evaluation of nasal histology, approximately 4 µm thick sections coronally sectioned from the nasal vestibule were stained with Hematoxylin and Eosin (H&E). The number of eosinophils in the nasal mucosa was counted under a light microscope (×400 magnification).

All data were analyzed with GraphPad Prism software (version 8.3; GraphPad Software, Inc., La Jolla, CA, USA) and are expressed as the mean ± Standard Deviation (SD). Correlations among the four groups were calculated using one-way analysis of variance followed by Tukey’s multiple comparison post hoc tests, p < 0.05 was considered statistically significant.

The main clinical features of healthy controls and AR patients are shown in Table 1. In our results, the expression level of miR-223-3p in AR serum was significantly higher than that in healthy control subjects (Fig. 2A). Receiver operating characteristic (ROC) curves were generated for miR-223-3p expression, and the sensitivity and specificity were calculated based on the ROC curves. The sensitivity and specificity of serum miR-223-3p were determined by constructing an ROC curve with an Area Under the Curve (AUC) of 0.845, revealing a relatively high diagnostic power in distinguishing AR patients from healthy controls (Fig. 2B). These results collectively imply that miR-223-3p might be a potential positive regulator of the pro-allergic factors of AR and a promising clinical biomarker in patients with AR.

Figure 2.

MiR-223-3p is highly expressed and serves as a clinical biomarker in AR patients. (A) The expression level of miR-223-3p in AR patients (n = 41) was significantly higher than that in healthy participants (n = 39). (B) ROC curve analysis of the diagnosis of AR for the miR-223-3p marker. AUC: area under the curve. Statistical significance was assessed using two-tailed Student’s t-test. The error bar indicates the SD. (****p < 0.0001).

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To investigate the effects of miR-223-3p on the allergic response in AR, we overexpressed or downregulated miR-223-3p in the nasal mucosa of AR mice by intranasal administration of miR-223-3p agomir or miR-223-3p antagomir. As shown in Fig. 3, miR-223-3p expression was significantly elevated in AR mice compared with the control group and even increased slightly in miR-223-3p agomir-treated AR mice. In contrast, miR-223-3p expression was much lower in mice after nasal administration of miR-223-3p antagomir than in AR mice. These results suggest that miR-223-3p might facilitate AR and provide evidence for successful gene transduction in these mice.

Figure 3.

Expression of miR-223-3p in nasal mucosa of AR mice following intranasal administration of mismatched agomir/miR-223-3p agomir/mismatched antagomir/miR-223-3p antagomir. AR mice were induced as described in a previously published protocol and then intranasally treated with mismatched agomir/miR-223-3p agomir/mismatched antagomir/miR-223-3p antagomir after the last OVA challenge for one week. The expression of miR-223-3p in nasal mucosa was determined by qRT-PCR. (**p < 0.01, ****p < 0.0001).

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We also evaluated the effects of miR-223-3p on the allergic response in AR mice by measuring OVA-specific IgE in the serum of these mice. The concentration of OVA-specific IgE was significantly increased in AR mice compared with normal mice (Fig. 4A). Furthermore, upregulation of miR-223-3p levels via intranasal administration of miR-223-3p agomir increased the OVA-specific IgE concentration, which was much lower in the miR-223-3p antagomir-treated group (Fig. 4 B,C). In addition, the nasal allergic symptoms, including the occurrences of nasal rubbing and sneezing, were counted within 30 min of the last OVA challenge on day 34. As displayed in Fig. 4D, the symptom score increased markedly in AR mice compared with normal mice and even became significantly higher in the miR-223-3p agomir-treated AR mice. The symptom score appeared to decrease in mice treated with miR-223-3p antagomir.

Figure 4.

Effects of miR-223-3p on OVA-specific IgE and allergic symptoms in AR mice. (A) OD values of OVA-specific IgE in the serum of both the control and AR groups were measured using ELISA. (B) OD values of OVA-specific IgE in serum of the mismatched agomir and miR-223-3p agomir groups were measured using ELISA. (C) OD values of OVA-specific IgE in serum of the mismatched antagomir and miR-223-3p antagomir groups were measured using ELISA. (D) The symptom scores of the above three groups were calculated. The error bar indicates the SD. (*p < 0.05, **p < 0.01, ***p < 0.001).

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A previous study reported that intranasal administration of the OVA antigen could induce a Th2 lymphocyte response in the nasal mucosa, which generated IL-5 through the activation of innate lymphoid cells.23 Thus, we next investigated whether the upregulation of miR-223-3p increases Th2 immunity to OVA in vivo. Not surprisingly, the nasal mucosa tissues of AR mice produced higher Th2 cytokines (IL-4, IL-5) and higher Th1 cytokines (IFN-γ). Furthermore, all these cytokine levels increased in miR-223-3p agomir-treated AR mice compared with untreated AR mice (Fig. 5A,B, D,E, G,H). The mRNA expression levels of IL-4, IL-5 and IFN-γ were attenuated by miR-223-3p antagomir administration (Fig. 5 C, F and I).

Figure 5.

MiR-223-3p increases cytokine levels in AR mice. The mRNA expression levels of IL-4 (A‒C), IL-5 (D‒F), and IFN-γ (G‒I) were detected by qRT-PCR. Statistical significance was assessed using two-tailed Student’s t-test. The error bar indicates the SD. (*p < 0.05, **p < 0.01, ****p < 0.0001).

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To further confirm the effect of miR-223-3p on the pathological alterations in the nasal mucosa of AR mice, paraffin-embedded tissue sections were examined using H&E staining. Consistent with the above results, OVA sensitization induced apparent pathological changes in nasal mucosal epithelium. Significantly worse hyperemia, edema, and chaotic structure existed in the nasal mucosal epithelium of AR mice than in normal control mice (Fig. 6 A,B). Furthermore, all these pathological alterations were aggravated after miR-223-3p agomir administration (Fig. 6C), and miR-223-3p antagomir ameliorated the allergic inflammation (Fig. 6D). To explore the effect of miR-223-3p on the infiltration of inflammatory cells into the nasal mucosa of AR, eosinophil cells were subsequently counted. We observed significantly more eosinophil cells in the nasal mucosa in AR mice than in control mice. Furthermore, intranasal administration of miR-223-3p agomir markedly increased the number of eosinophil cells, which was reduced in the miR-223-3p antagomir-treated group (Fig. 6E). These data demonstrated that miR-223-3p could promote allergic pathology with immune cell infiltration in the nasal mucosa of AR mice.

Figure 6.

MiR-223-3p promoted an allergic inflammatory response in the nasal mucosa of AR mice. (A–D) The pathological changes in nasal mucosa from each group were analyzed using H&E staining. (E) The number of eosinophils in each group was quantified. (*p < 0.05, **p < 0.01, ***p < 0.001.

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To further investigate the mechanisms by which miR-223-3p promoted allergic responses and symptoms in AR mice, potential target genes of miR-223-3p were predicted using the miRNA target gene prediction databases TargetScan (http://www.targetscan.org/vert_71/), miRWalk (http://mirwalk.umm.uni-heidelberg.de/) and miRDB (http://www.mirdb.org/). Notably, it was predicted that miR-223-3p might directly target inositol polyphosphate 4 phosphatase (INPP4A), a gene known to be involved in asthma and allergic disorders.24 As shown in Fig. 7A, an 8-bp fragment of the 3′UTR of the INPP4A gene is complementary to the miR-223-3p seed sequence. To verify this prediction, successful overexpression or knockdown of miR-223-3p in HEK293 T cells was verified by quantitative real-time PCR. As shown in Fig. 7B, overexpression of miR-223-3p by transfection of miR-223-3p mimics markedly decreased the mRNA expression of INPP4A, which was remarkably restored via transduction of miR-223-3p inhibitor. These results suggest that miR-223-3p may promote allergic inflammatory responses by negatively regulating INPP4A expression.

Figure 7.

MiR-223-3p negatively regulates INPP4A expression. (A) The schematic of INPP4A mRNA shows a potential site in the INPP4A 3′-UTR for miR-223-3p. (B) qRT-PCR analysis of INPP4A mRNA levels in HEK293T cells transfected with miR-223-3p mimic or inhibitor. Statistical significance was assessed using Tukey’s multiple comparison post hoc test. The error bar indicates the SD. (*p < 0.05).

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