A human gastric cancer cell line, SGC-7901 was purchased from the cell bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). The cells were routinely cultured in RPMI-1640 medium purchased from Gibco supplemented with 10% FBS (both Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C in a humidified atmosphere of 5% CO₂. H. pylori strain 26695 was obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in brain-heart infusion agar (BHI; Becton, Dickinson and Company, Franklin Lakes, NJ, USA) plates with 10% rabbit blood (cat. no. HQ50073; Shanghai Bangsheng Biotechnology Co., Ltd., Shanghai, China) at 37°C under the microaerophilic conditions of 5% O₂, 10% CO₂ and 85% N₂.

H. pylori was collected from the culture plates using 2 ml sterile PBS followed by centrifugation at 2500 × g for 5 min at room temperature, and resuspended in RPMI-1640 medium. The concentration of H. pylori 26695 was determined at 600 nm using the Agilent 8453E UV-visible Spectroscopy System (Agilent Technologies, Inc., Santa Clara, CA, USA): Optical density (600 nm) = 1×10⁹ colony forming unit/ml. SGC-7901 cells were infected with H. pylori 26695 for 6 h at 37°C, with the multiplicity of infection 100.

An ELISA was employed to evaluate the inflammatory response of SGC-7901 cells subjected to IL-17A stimulation at serial concentrations (0, 0.01, 0.1, 1 or 10 ng/ml) over 24 h at 37°C, or treated with 10 ng/ml IL-17A at different time points (0, 6, 12, 18 or 24 h) at 37°C. Cell culture supernatants were collected by centrifugation at 1,000 × g for 20 min at room temperature, and the expression levels of IL-17A (cat. no. D1700), GRO-α (cat. no. DGR00B) and IL-8 (cat. no. D8000C) in the supernatant were determined using DuoSet ELISA Development System (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's protocols. Absorbance at 450 nm was detected using a microplate reader.

Total RNA was extracted from tissues or cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. For RT-qPCR analysis of miR-146a, the total RNA isolated from cells was reverse transcribed to cDNA using a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) The reactions were incubated at 16°C for 30 min, followed by 42°C for 30 min, then 85°C for 5 min before being held at 4°C. The expression levels of miR-146a were evaluated using TaqMan miRNA assays (Ambion; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. The reactions were performed using a Bio-Rad IQ5 (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with the following program: 95°C for 2 min, 40 cycles of 95°C for 15 sec and 60°C for 30 sec. U6 was used as internal reference. The upstream and downstream primers of miR-146a and U6 were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) and the sequences were as follows: miR-146a forward, 5′-GTGCAGGGTCCGAGGT-3′ and reverse, 5′-CGGCGGTGAGAACTGAATTCC-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′. Relative expression of miR-146a was analyzed using 2^−∆∆Cq method (27). The results were repeated at least three times.

The expression levels of IL-17A were determined using a PrimeScript RT-PCR kit (Takara Bio, Inc., Otsu, Japan). The expression was normalized using β-actin. The primer sequences are as follows: IL-17A forward, 5′-AGGAATCACAATCCCACGAA-3′ and reverse, 5′-ACTTTGCCTCCCAGATCACA-3′; β-actin forward, 5′-TTCCTTCCTGGGCATGGAGTCC-3′ and reverse, 5′-TGGCGTACAGGTCTTTGCGG-3′.

All oligonucleotides were obtained from Shanghai GenePharma Co., Ltd. (Shanghai, China) and transfected into SGC-7901 cells using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Briefly, SGC-7901 cells were seeded in 6-well plates at a concentration of 2.0×10⁵ cells/well in RPMI-1640 supplemented with 10% FBS. After subculturing at 37°C for 24 h under 5% CO₂, the medium was replaced with fresh serum-free medium purchased from Gibco (Opti-MEM^® I Reduced Serum Medium; Thermo Fisher Scientific, Inc.), and SGC-7901 cells were transfected with miR-146a mimics (50 nM; 5′-UGAGAACUGAAUUCCAUGGGUU-3′; 5′-CCCAUGGAAUUCAGUUCUCAUU-3′), scrambled miR control (50 nM; forward, 5′-UUCUCCGAACGUGUCACGUTT-3′ and reverse, 5′-ACGUGACACGUUCGGAGAATT-3′), IRAK1 small interfering (si)RNA (100 nM; forward, 5′-GGUUUCGUCACCCAAACAUtt-3′ and reverse, 5′-AUGUUUGGGUGACGAAACCtg-3′) or TRAF6 siRNA (100 nM; forward, 5′-GGUUGUUUGCACAAGAUGGtt-3′ and reverse, 5′-CCAUCUUGUGCAAACAACCtt-3′). The mock treatment group was transfected with transfection reagent only. FAM-labeled negative control siRNA (100 nM) purchased from Ambion (cat. no. AM4620; Thermo Fisher Scientific, Inc.) was transfected into SGC-7901 cells to evaluate the transfection efficiency; >80% of the cells were successfully transfected (data not shown). After transfection for 24 h, the cells were treated with 10 ng/ml IL-17A (cat. no. 200-17; Peprotech, Inc., Rocky Hill, NJ, USA) for 24 h.

Western blot analysis was performed as previously described (28). The blot was probed with primary antibodies at 4°C overnight: Anti-IRAK1 (1:1,000; cat. no. 4362) and anti-TRAF6 (1:1,000; cat. no. 4743), were used to determine the expression levels of IRAK1 and TRAF6, respectively; Anti-β-actin (1:1,000; cat. no. 5125; all Cell Signaling Technology, Inc., Danvers, MA, USA) was used as an internal control. Next, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies, goat-anti-mouse (1:5,000; cat. no. 115-035-003) or goat-anti-rabbit (1:5,000; cat. no. 111-035-003; both Jackson ImmunoResearch Laboratories., Inc., West Grove, PA, USA) at 37°C for 2 h. Protein bands were visualized using SuperSignal West Dura Duration substrate reagent (cat. no. 34080; Thermo Fisher Scientific, Inc.). Blots were quantified using Image J software (version 1.48; National Institutes of Health, Bethesda, MD, USA).

For NF-κB activity analysis, SCG-7901 cells were co-transfected with 0.8 mg of the reported luciferase vector pNF-κB-TA-Luc (cat. no. 631904; Clontech Laboratories, Inc., Mountainview, CA, USA), 0.04 mg of Renilla control vector (pRL-TK; cat. no. 2241; Promega Corporation, Madison, WI, USA) and miR-control/miR-146a mimics by using Lipofectamine 2000 according to the manufacturer's protocols. After 24 h, the cells were stimulated with or without 10 ng/ml IL-17A (cat. no. 200-17; Peprotech, Inc.) for 12 h at 37°C. Luciferase activity was analyzed using the Dual-Luciferase Reporter Assay System (Promega Corporation). Firefly luciferase activity was normalized to that of Renilla luciferase.

In total, 40 samples from were collected from patients with gastric mucosa (18 women and 22 men, 25–60-years-old) who underwent endoscopy due to dyspeptic symptoms at Southwest Hospital (Chongqing, China) between January 2013 and December 2014, including 20 patients with H. pylori–induced chronic gastritis and 20 healthy donors. None of the patients had been administered nonsteroidal anti-inflammatory drugs, antibiotics or proton pump inhibitor drugs. Identification of H. pylori infection in gastric mucosa was performed via H. pylori culture, C13-urea breath tests and histological analysis. The present study was approved by the Ethics Committee of the Institutional Review Board at The Third Military Medical University (Chongqing, China), and written informed consent was obtained from all patients.

Data were presented as the mean ± standard deviation of at least three independent experiments. Data were analyzed using a Student's t-test or analysis of variance followed by a Dunnett's post-hoc test. The association between the expression levels of miR-146a and IL-17A was analyzed using Pearson's correlation. All statistical analyses were performed using GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

Previous studies reported that H. pylori infection may stimulate the secretion of IL-17A (10,11,29), which was also confirmed in human gastric cancer cell line SGC-7901 in the present study. As presented in Fig. 1A, H. pylori infection significantly increased the protein levels of IL-17A in SGC-7901 cells compared with the control. Furthermore, to investigate the roles of IL-17A in inflammation, the expression levels of GRO-α and IL-8 were examined by ELISA. As presented in Fig. 1B, production of GRO-α and IL-8 were significantly increased in SGC-7901 cells following treatment with 0.01–10 ng/ml IL-17A over 24 h compared with the control. The expression levels of GRO-α and IL-8 in SGC-7901 cells were also significantly increased in a time-dependent manner (Fig. 1C). These data indicated that H. pylori-associated production of IL-17A could upregulate GRO-α and IL-8 in gastric epithelial cells, suggesting a potential role of IL-17A in H. pylori-induced inflammation.

Previous studies have revealed that the expression levels of miR-146a was significantly increased in gastric epithelial cells following the stimulation by H. pylori (20,30). Whether H. pylori-induced IL-17A can enhance the expression of miR-146a in SGC-7901 cells was further investigated in the present study. As presented in Fig. 2A, the expression levels of miR-146a were significantly increased in SGC-7901 cells treated with 1 and 10 ng/ml IL-17A over 12 h compared with the control; but no significant difference was observed in SGC-7901 cells treated with 0.01 or 0.1 ng/ml IL-17A. In addition, the expression of miR-146a was significantly upregulated from 6–24 h following treatment with 10 ng/ml IL-17A compared with the control; expression peaked at 12 h and reduced thereafter (Fig. 2B). These results suggested that miR-146a expression is increased in gastric epithelial cells upon the stimulation with IL-17A.

A previous study indicated that IL-17A induced the production of proinflammatory cytokines via the NF-κB signaling pathway (31). Thus, whether miR-146a regulated proinflammatory cytokines in a NF-κB-dependent manner was investigated. As presented in Fig. 3A, luciferase report assays revealed that miR-146a mimics significantly inhibited the activity of NF-κB compared with the control. Furthermore, IRAK1 and TRAF6 have been reported as two potential targets of miR-146a, and are involved in the activation of NF-κB (30). In the present study, miR-146a significantly reduced the protein expression of IRAK1 and TRAF6 upon stimulation with IL-17A (Fig. 3B). To further determine the biological functions of IRAK1 and TRAF6 on IL-17A-stimulated inflammatory responses, specific siRNAs were used to inhibit the expression of IRAK1 and TRAF6 (Fig. 3C). Western blot analysis revealed that downregulation of IRAK1 and TRAF6 significantly decreased the protein levels of GRO-α and IL-8 compared with the control (Fig. 3D), which suggested that IL-17A-induced expression of miR-146a may suppress the expression of IRAK1 and TRAF6, attenuating NF-κB activity and affecting inflammation.

Previous studies revealed that miR-146a participated in the negative feedback regulation of inflammation during H. pylori infection (20–22,30); thus the effects of miR-146a on IL-17A-induced inflammatory responses in SGC-7901 cells were investigated. The expression levels of miR-146a in transfected SGC-7901 cells were determined using RT-qPCR (Fig. 4A). As presented in Fig. 4B, SGC-7901 cells were transfected with miR control or miR-146a mimics followed by treatment with IL-17A. As presented in Fig. 4B, miR-146a mimics significantly decreased the protein levels of GRO-α and IL-8 compare with the control. In summary, these data suggested that miR-146a serves a potential role in IL-17A-induced inflammatory responses.

To determine whether the expression levels of IL-17A were increased in H. pylori-infected gastric mucosa in vivo, the mRNA expression of IL-17A in gastric mucosa tissues from patients with H. pylori infection was determined. The mRNA levels of miR-146a and IL-17A were evaluated in gastric mucosa tissues from 20 patients with H. pylori-induced chronic gastritis and 20 healthy donors. The results of RT-qPCR revealed that the expression levels of IL-17A and miR-146a (Fig. 5A and B) were upregulated in H. pylori-infected gastric mucosa tissues compared with the normal tissues. Furthermore, Pearson's correlation analysis indicated a positive correlation between the mRNA levels of miR-146a and IL-17A (R²=0.5829, P<0.0001; Fig. 5C), suggesting that miR-146a is correlated with the production of IL-17A in H. pylori-infected human gastric mucosa.