Nasal mucosa samples from the inferior turbinate mucosa were obtained from 20 healthy subjects (13 males and 7 females; age, 32.1±16.0) and 20 patients with AR. (males and 9 females; age, 36.4±13.8). Samples were stored at −80°C until further use. The present study was approved by The Ethics Committee of The Shanghai Ninth People's Hospital (Shanghai, China). Written informed consent was obtained from all patients.

Under local anesthesia, inferior turbinate nasal epithelial cells (NECs) were obtained using the nasal fragment method (20). Cells were cultured in bronchial epithelial cell growth medium (BEGM; Lonza Group, Ltd.) with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) in a humidified 5% CO₂ incubator. The NECs were passaged when a 70–80% fused monolayer was present. Cells from passage 2 were used for subsequent experiments.

Small interfering RNA (siRNA) targeting circARRDC3 (si-circARRDC3) and negative control (si-NC), miR-375 mimic and negative control (NC mimic), and miR-375 inhibitor and negative control (NC inhibitor) were purchased from Shanghai GenePharma Co., Ltd. circARRDC3 or KLF4 cDNA was amplified and subcloned into pcDNA3.1 vectors (Shanghai GenePharma Co., Ltd.) to overexpress circARRDC3 or KLF4. pcDNA3.1 empty vector (pcDNA3.1) as a control.

All plasmids (0.5 µg) and oligonucleotides (50 nM) were transfected into NECs cells (6×10⁵ cells/well) using Lipofectamine™ 2000 (Thermo Fisher Scientific, Inc.). After transfection for 48 h, the cells were used for subsequent assays. The sequences of the oligonucleotides used for transfection were as follows: i) si-circARRDC3, 5′-CAGACCTCATCACTCAGAA-3′; ii) siNC, 5′-CCGAAGGCCGTGTCCCGAG-3′; iii) miR-375 mimic, 5′-UUUGUUCGUUCGGCUCGCGUGA-3′; iv) NC mimic, 5′-UUUGUACUACACAAAAGUACUG-3′; v) miR-375 inhibitor, 5′-UCACGCGAGCCGAACGAACAAA-3′; and vi) NC inhibitor, 5′-CAGUACUUUUGUGUAGUACAAA-3′.

Following lentivirus infection, NECs (0.75×10⁵/well) were stimulated with 50 ng/ml IL-13 (R&D Systems, Inc.) for 14 days at 37°C in BEGM without hydrocortisone. The media was replaced twice per week. Cell supernatant and pellets were collected centrifuged for 3 min at 4°C for reverse transcription-quantitative PCR (RT-qPCR).

Transfected cells were seeded into 96-well culture plates (2×10⁴ cells/well). A total of ~20 µl MTT solution was added to each well, and the cell culture plates were incubated at 37°C for 4 h. DMSO (150 µl) was added following supernatant removal. The absorbance was determined at a wavelength of 490 nm using a microplate reader (Bio-Rad Laboratories, Inc.).

The Starbase (version 2.0; http://starbase.sysu.edu.cn/starbase2/) and TargetScan (Release 7.2; http://www.targetscan.org/vert_72/) online tools were used to predict the downstream targets of circARRDC3 and miR-375, respectively (21). Wild-type and mutant circARRDC3 or KLF4 sequences were subcloned into the pmirGLO vector (Promega Corporation). Subsequently, the vectors were co-transfected with miR-375 mimic, NC mimic, miR-375 inhibitor or NC inhibitor into 293T cells (2×10⁵ cells/well) using Lipofectamine^® 2000 (Thermo Fisher Scientific, Inc.). Following 48 h of transfection, a dual-luciferase reporter system (Promega Corporation) was used to measure luciferase activity. Firefly luciferase activity was normalized to Renilla luciferase.

Total RNA was extracted from NECs or nasal mucosal specimens using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). RT was performed using the PrimeScript Reagent kit (Takara Bio, Inc.) at 37°C for 30 min. qPCR was performed using the SYBR-Green PCR Master Mix kit (Takara Bio, Inc.) on the ABI Prism 7300 Sequence Detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions consisted of an initial denaturation at 95°C for 15 sec, followed by 40 cycles of denaturation at 94°C for 30 sec, annealing at 60°C for 20 sec, and extension at 72°C for 40 sec. GAPDH and U6 were used as internal controls. Gene expression was analyzed using the 2^−∆∆Cq method (22). The following primer pairs were used for the qPCR: circARRDC3 forward, 5′-TCCTATTGCTCTTCCTTGTGG-3′ and reverse, 5′-AAAAAGTCAGGGCAGCAGAG-3′; miR-375 forward, 5′-AAACCGGACCTGAGCGTTTT-3′ and reverse, 5′-CCGAACGAACAAAACGCTCA-3′; KLF4 forward, 5′-TCTCCCACATGAAGCGACTT-3′ and reverse, 5′-ATGGGTCAGCGAATTGGAGA-3′; GAPDH forward, 5′-TCGTGGAAGGACTCATGACC-3′ and reverse, 5′-ATGATGTTCTGGAGAGCCCC-3′; and U6 forward, 5′-CTCGCTTCGGCAGCACATATACTA-3′ and reverse, 5′-ACGAATTTGCGTGTCATCCTTGCG-3′; GM-CSF forward, 5′-ATGTGGCTGCAGAGCCTGCTGC-3′ and reverse, 5′-CTCCCAGCAGTCAAAGGG-3′; eotaxin forward, 5′-CCCCTTCAGCGACTAGAGAG-3′ and reverse, 5′-TCTTGGGGTCGGCACAGAT-3′; MUC5AC forward, 5′-TGATCATCCAGCAGGGCT-3′ and reverse, 5′-CCGAGCTCAGAGGACATATGGG-3′.

NECs were lysed with pre-cooled RIPA lysis buffer (Beyotime Institute of Biotechnology) and quantified using a BCA Protein Assay kit (Beyotime Institute of Biotechnology). A total of 20 µg protein/lane was added into each well of a vertical electrophoresis tank and separated by SDS-PAGE on 10% gels. Subsequently, the proteins were transferred to PVDF membranes. After blocking with 5% skim-milk for 1 h at room temperature, the membranes were incubated with primary antibodies specific for KLF4 (1:1,000; Novous; cat. no. IMG-6081A) and GAPDH antibody (1:500; Santa Cruz Biotechnology, Inc.; cat. no. sc-47724) at 4°C overnight. The membranes were washed three times with 0.1% TBS-Tween-20 (each time 5 min), then incubated with goat anti-rabbit/mouse HRP-conjugated secondary antibodies (1:5,000; Abcam; cat. nos. ab205718 and ab205719) at room temperature for 1 h. Protein bands were visualized using the Enhanced Chemiluminescence Plus kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. Protein bands were quantified using Quantity One software (version 4.6.6; Bio-Rad Laboratories, Inc.).

The TUNEL assay was performed using an In Situ Cell Death Detection kit (Roche Diagnostics GmbH) according to the manufacturer's instructions. After rising twice by PBS, NECs cells fixed by 4% formaldehyde for 25 min at 4°C. The cells were permeabilized by 0.2% Triton X-100 for 5 min. The cells were then equilibrated with 100 µl Equilibration buffer for 10 min at room temperature. Subsequently, cells were labeled with 50 µl terminal deoxynucleotidyl transferase (TdT) reaction cocktail for 45 min at 37°C, followed by treatment with Click-iT reaction cocktail. The nucleus was stained with hematoxylin or methyl green at room temperature for 1 h. A fluorescence microscope (magnification, ×100; Olympus Corporation) was utilized to observe TUNEL-positive cells in at least 5 fields of view.

Statistical analyses were performed using SPSS 23.0 software (IBM Corp.). Data are presented as the mean ± SD from at least three independent experiments. Student's t-test or one-way ANOVA followed by Tukey's post hoc test were used for comparisons between groups. Correlation analysis was carried out using Pearson's correlation coefficient. P<0.05 was considered to indicate a statistically significant difference.

To determine the effects of circARRDC3 on AR development, si-circARRDC3 or pcDNA3.1-circARRDC3 were transfected into NECs to inhibit or overexpress circARRDC3 (Fig. 1A). As shown in Fig. 1B-D, knockdown of circARRDC3 downregulated the levels of GM-CSF, eotaxin and MUC5AC, whereas circARRDC3 overexpression upregulated the levels of GM-CSF, eotaxin and MUC5AC. In addition, circARRDC3 interference significantly promoted the viability of IL-13-treated NECs, whereas circARRDC3 overexpression significantly repressed cell viability (Fig. 1E). Furthermore, knockdown of circARRDC3 significantly suppressed the apoptosis of IL-13-treated NECs, and the addition of circARRDC3 significantly promoted cell apoptosis (Fig. 1F). Collectively, the data indicated that circARRDC3 may contribute to AR development.

Based on bioinformatics analysis (StarBase 2.0), miR-375 was predicted to be a downstream target of circARRDC3 (Fig. 2A). RT-qPCR indicated that the expression of miR-375 was upregulated in NECs transfected with miR-375 mimic and was downregulated in NECs transfected with miR-375 inhibitor (Fig. 2B). As shown in Fig. 2C, transfection with the miR-375 mimic decreased and miR-375 inhibitor increased the luciferase activity of circARRDC3-WT, while no significant difference was observed in the circARRDC3-Mut group. To determine whether miR-375 was critical for circARRDC3-mediated AR development, si-NC, si-circARRDC3 and si-circARRDC3 + miR-375 inhibitor were transfected into IL-13-treated NECs. RT-qPCR revealed that knockdown of circARRDC3 significantly upregulated miR-375 expression, which was reversed following miR-375 inhibitor transfection (Fig. 2D). Moreover, transfection with the miR-375 inhibitor reversed the inhibitory effects of circARRDC3 knockdown on the levels of GM-CSF, eotaxin and MUC5AC in IL-13-induced NECs (Fig. 2E-G). Further results indicated that circARRDC3 knockdown promoted the viability of IL-13-induced NECs, whereas the effects were reversed by miR-375 inhibition (Fig. 2H). Furthermore, the depletion of circARRDC3 suppressed the apoptosis of IL-13-induced NECs, which was abolished following miR-375 inhibitor transfection (Fig. 2I). In summary, these results revealed that circARRDC3 regulated AR progression by targeting miR-375 in IL-13-induced NECs.

To investigate the potential effects of miR-375 in NECs, bioinformatics tools (TargetScan) were used to predict the possible targets. It was found that the 3′-UTR of KLF4 had direct binding sites for miR-375 (Fig. 3A). In addition, the luciferase reporter assay indicated that transfection with the miR-375 mimic decreased and the miR-375 inhibitor increased the luciferase activity of KLF4-WT, while there were no changes observed in the KLF4-Mut group (Fig. 3B). Moreover, RT-qPCR and western blotting revealed that miR-375 upregulation decreased KLF4 expression and miR-375 downregulation increased KLF4 expression (Fig. 3C and D). Collectively, the results suggested that miR-375 could directly interact with KLF4 and significantly inhibit its expression.

To further assess the roles of KLF4 and miR-375 in AR, NC mimic, miR-375 mimic, and miR-375 mimic + KLF4 were transfected into IL-13-induced NECs. RT-qPCR showed the KLF4 expression was significantly increased in NECs transfected with pcDNA3.1-KLF4 (Fig. 3E). As shown in Fig. 3F-H, KLF4 overexpression reversed the suppressive effects of miR-375 mimic transfection on the expression levels of GM-CSF, eotaxin and MUC5AC. In addition, the upregulation of miR-375 promoted cell viability in IL-13-induced NECs, while these effects were abolished by KLF4 overexpression (Fig. 3I). Furthermore, miR-375 mimic inhibited apoptosis of IL-13-induced NECs, which was partially reversed following KLF4 overexpression (Fig. 3J). The results indicated that miR-375 expression negatively regulated KLF4 expression by directly interacting with each other in IL-13-induced NECs.

RT-qPCR showed that circARRDC3 and KLF4 were increased, and miR-375 was decreased in AR tissues (Fig. 4A-C). Furthermore, miR-375 expression was negatively correlated with circARRDC3 or KLF4 expression. Whereas, circARRDC3 expression was positively correlated with KLF4 expression in AR tissues (Fig. 4D-F). To summarize, circARRDC3, miR-375 and KLF4 were dysregulated in the clinical nasal mucosa of patients with AR.