A total of 16 children (8 male and 8 female; mean age ± standard deviation, 11.4±2.16 years; age range, 8–15) with pneumonia were recruited to the present study. Pneumonia was diagnosed according to clinical presentation, respiratory symptoms, bacterial infection and chest X-ray. In addition, 16 gender-matched children (mean age ± standard deviation, 11.3±2.24 years; age range, 7–15) with fever were included as the control group; these children were treated in the Emergency Department and had no respiratory symptoms. These 32 patients were recruited from the Jinan Maternity and Child Care Hospital (Jinan, China) between June 2012 and December 2013. None of the patients underwent any treatment prior to blood collection. Venous blood samples (5 ml) were collected from the patients and the present study was approved by the Ethics Committee of the Jinan Maternity and Child Care Hospital. Written informed consent was obtained from the legal guardians of all participants.

The A549 human lung adenocarcinoma cell line was purchased from the Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI 1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (both Beijing Transgen Biotechnology Co., Ltd., Beijing, China) at 37°C in an incubator containing 5% CO₂. A total of 2 ml cells/well, at a concentration of 5×10⁵ cells/ml, were cultured in a 6-well plate. Subsequently, cells were separated into groups, and were treated with LPS (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), miR-20a mimic (sense 5′-UAAAGUGCUUAUAGUGCAGGUAG-3′ and antisense 5′-ACCUGCACUAUAAGCACUUUAUU-3′), mimic control (sense 5′-UGCUUAGUAUAAGAUGCAGGUAG-3′ and antisense 5′-ACGCUAUAUCUUAUAGCACACUU-3′) (both Shanghai GenePharma Co., Ltd., Shanghai, China) or the nuclear factor (NF)-κB inhibitor pyrrolidine dithiocarbamate (PDTC; Sigma-Aldrich; Merck KGaA). Briefly, the cells were divided into the following four groups: Control group (non-treated cells); LPS group, in which cells were stimulated with LPS (1 µg/ml) for 12 h at 37°C to induce inflammatory lung cell injury (20); LPS + miR-20a group, in which cells overexpressing miR-20a were treated with LPS (1 µg/ml) for 12 h at 37°C; and LPS + miR-20a + PDTC group, in which cells overexpressing miR-20a were treated with PDTC (100 mmol/l) for 30 min at 37°C and then treated with LPS (1 µg/ml) for 12 h at 37°C (21). In addition, an LPS + mimic control group, in which cells were transfected with mimic control and then treated with LPS (1 µg/ml) for 12 h at 37°C, was generated. To overexpress miR-20a, the miR-20a mimic (50 nM) was transfected into A549 cells (1×10⁵ cells) using 20 µl Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. Transfection with the mimic control was the same as the transfection of miR-20a. Transfected cells were harvested at 48 h post-transfection for subsequent experiments.

Blood samples were collected from the children and were centrifuged at 1,500 × g for 10 min at 4°C. The supernatant serum samples were then collected and stored at −80°C. The serum concentrations of interleukin (IL)-6 (cat. no. S6050), tumor necrosis factor (TNF)-α (cat. no. STA00C) and C-reactive protein (CRP; cat. no. SCRP00) were determined using corresponding ELISA kits (R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer's protocols. The absorbance was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, Inc.). The concentrations of IL-6, TNF-α and CRP were calculated based on the standard curve.

Total RNA was extracted from blood samples using the RNAprep pure Blood kit (Tiangen Biotech, Beijing, China) and total RNA was extracted from treated cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Subsequently, RNA was measured using a UV spectrophotometer (BD Biosciences, San Diego, CA, USA) at A₂₆₀, and cDNA was generated from miRNAs using stem-loop RT primers (Applied Biosystems; Thermo Fisher Scientific, Inc.) with the following cycling parameters: 30°C for 10 min, followed by 42°C for 30 min and 95°C for 5 min. Primer sequences for miR-20a and U6 small nuclear (sn)RNA are presented in Table I. SYBR^® Premix Ex Taq^™ kit (Takara Biotechnology Co., Ltd., Dalian, China) was used to perform PCR amplification; the PCR program was as follows: 94°C for 10 min, followed by 40 cycles at 94°C for 15 sec, 56°C for 30 sec and 72°C for 1 min, and finally 72°C for 10 min. U6 snRNA was used as an internal control. Relative quantification and calculations were conducted using the quantification cycle (Cq) method (2^−ΔΔCq) (22).

The cells were collected and the proteins were extracted using RIPA buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing a cocktail of protease inhibitors (Roche Diagnostics, Basel, Switzerland). Bicinchoninic acid protein assay kit (Sangon Biotech Co., Ltd., Shanghai, China) was used to determine protein concentration. Subsequently, equal amounts of protein (30 µg/lane) were separated by 10% SDS-PAGE and were transferred to a polyvinylidene fluoride membrane. After blocking with 5% non-fat milk at room temperature for 2 h, the membrane was incubated with rabbit anti-human inhibitor of NF-κB α (IκBα; cat. no. SAB4501994; 1:500), phosphorylated (p)-NF-κB (cat. no. SAB4301496; 1:500) polyclonal antibodies and β-actin monoclonal antibody (cat. no. SAB2100037; 1:1,000) (all Sigma-Aldrich; Merck KGaA) at 4°C overnight. β-actin was used as an internal control. After washing with PBS, the membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (cat. no. 31460; 1:10,000; Thermo Fisher Scientific, Inc.) for 2 h at room temperature. The membrane was washed again with PBS and color was developed using a 3,3′-diaminobenzidine substrate (Sangon Biotech Co., Ltd.). Image Pro Plus software (version 5.0; Media Cybernetics, Inc., Rockville, MD, USA) was used to semi-quantify the expression levels of the target proteins.

All data are presented as the mean ± standard deviation, and SPSS 16.0 statistics software (SPSS, Inc., Chicago, IL, USA) was used to analyze the data. All data were initially tested for Gaussian distribution. Subsequently, data were analyzed by unpaired two-tailed t-test (for two groups) or one-way analysis of variance followed by a Tukey post hoc test (for more than two groups). P<0.05 was considered to indicate a statistically significant difference.

To explore the expression levels of miR-20a in patients with pneumonia, an RT-qPCR analysis was performed; the results demonstrated that miR-20a expression was higher in patients with pneumonia compared with in healthy controls (P<0.01; Fig. 1A). In addition, LPS was used to induce inflammatory injury in A549 cells. Compared with in the control group, the expression levels of miR-20a were increased in the LPS group (P<0.01; Fig. 1B).

The present study investigated the association between miR-20a and the inflammatory reaction. ELISA analysis revealed that the serum concentrations of inflammatory factors, including IL-6, TNF-α and CRP, were higher in patients with pneumonia compared with in healthy controls (P<0.05; Fig. 2A). The results of an RT-qPCR analysis indicated that the expression levels of miR-20a were significantly upregulated following transfection of cells with miR-20a mimic compared with in the mimic control group (P<0.001; Fig. 2B), confirming that miR-20a was successfully overexpressed. Subsequent experiments demonstrated that the concentrations of inflammatory factors, including IL-6, TNF-α and CRP, were increased in the LPS group compared with in the control group (P<0.05; Fig. 2C-E). In addition, overexpression of miR-20a further increased the concentrations of IL-6, TNF-α and CRP compared with the LPS group (P<0.05). The levels of inflammatory cytokines in the LPS + mimic control group were not significantly different compared with the LPS group (data not shown).

To further investigate the mechanism underlying the effects of miR-20a on the induction of inflammation, NF-κB signaling pathway-associated proteins, including IκBα and p-NF-κB, were measured by western blotting. As shown in Fig. 3A-C, the protein expression levels of IκBα and p-NF-κB were increased in the LPS group compared with in the control group (P<0.05). The expression of total NF-κB was consistent with that of β-actin (data not shown). In addition, the expression levels of IκBα and p-NF-κB were further increased in the LPS + miR-20a group compared with in the LPS group (P<0.05). The expression levels of IκBα and p-NF-κB in LPS + mimic control group were not significantly different compared with the LPS group (data not shown).

To evaluate the role of the NF-κB signaling pathway in miR-20a-induced inflammation, the NF-κB inhibitor PDTC was used to suppress the NF-κB signaling pathway. ELISA was subsequently used to detect the expression levels of inflammatory factors. The results demonstrated that following treatment with the NF-κB inhibitor PDTC, the expression levels of IL-6, TNF-α and CRP were markedly decreased compared with in the LPS + miR-20a group (P<0.05; Fig. 4). The levels of inflammatory cytokines in the LPS + mimic control group were not significantly different compared with the LPS group (data not shown).