The human acute monocytic leukemia THP-1 cell line was purchased from the Cell Bank of the Chinese Academy of Sciences. THP-1 cells were cultured using RPMI-1640 culture medium supplemented with 10% fetal bovine serum (both Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 mg/ml streptomycin at 37°C and 5% CO₂. Cells in the logarithmic growth phase were seeded into 6-well plate (1×10⁶ cells/well) and incubated overnight. They were subsequently transfected with 50 nM miR-146a mimic (cat. no. miR 10000449), 50 nM negative control (NC; cat. no. miR 01101) mimic, 100 nM miR-146a inhibitor or (cat. no. miR 20000449) 100 nM inhibitor NC (cat. no. miR 02101). All from Guangzhou RiboBio Co., Ltd.) using Lipofectamine^®2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. For the blank control, corresponding quantities of Opti-MEM (Gibco; Thermo Fisher Scientific, Inc.) solution were used in place of RNA during transfection (15).

At 24 h following transfection, THP-1 cells were washed with Hank's balanced salt solution (Beijing Solarbio Science & Technology Co., Ltd.) before the culture medium was replaced with fresh RPMI-1640 medium. A final concentration of 1 µg/ml LPS (Sigma-Aldrich; Merck KGaA) was subsequently added into the culture medium, followed by incubation for a further 3 h. The cells were then collected for the measurement of IRAK1 and p68 expression, whilst the supernatant of the culture medium was collected for the measurement of cytokine levels, as described previously (15). The cells were divided into the following experimental groups: Blank control, no RNA transfection or LPS treatment; LPS group, treated with LPS only; mimic group, miR-146 amimic+LPS; NC mimic group, NC mimic + LPS; inhibitor group, miR-146a inhibitor + LPS; and inhibitor NC group, inhibitor NC + LPS.

For the measurement of miRNA expression, small RNA was extracted from cells using the mirVana PARIS kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). cDNA was then reverse transcribed from small RNA using the TaqMan^® MicroRNA RT kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to manufacturer's protocol. The thermocycling conditions were as follows: 16°C for 30 min, 42°C for 30 min, 85°C for 5 min and 4°C hold. miR-146a expression levels were measured using TaqMan^® MicroRNA Assay kit (Assay ID, 000468; Applied Biosystems; Thermo Fisher Scientific, Inc.), the primers, which are patented, were provided by the manufacturer as part of the kit. U6 expression were used for the normalization of miRNA expression.

To determine mRNA expression levels, total RNA was extracted from cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and then reverse transcription was performed using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to manufacturer's protocol. The thermocycling conditions used was as follows: 25°C for 10 min, 37°C for 120 min, 85°C for 5 min and 4°C hold. qPCR was performed to measure IRAK1 mRNA levels in cells using PowerUp™ SYBR™ Green Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.). GAPDH was used as an internal reference. The sequences of the primers used were as follows: IRAK1 forward, 5′-GTGCTAGAGACCTTGGCTG-3′ and reverse, 5′-TGTGCTCTGGGTGCTTCTC-3′; and GAPDH forward, 5′-CCATGTTCGTCATGGGTGT-3′ and reverse, 5′-TGAGTCCTTCCACGATACC-3′.

Both qPCR assays aforementioned were performed using ABI Real Time PCR System 7500 (Applied Biosystems; Thermo Fisher Scientific, Inc.) using the following thermocycling protocol: Initiation reaction at 50°C for 2 min and 10 min at 95°C, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. The relative expression levels of miR-146a and IRAK1 were determined using the 2^−ΔΔCq method (16).

Following treatment with LPS for 3 h, the cells were collected and total protein was extracted using RIPA buffer (Pierce; Thermo Fisher Scientific, Inc.) supplemented with a cocktail of protease and phosphatase inhibitors (Sigma-Aldrich; Merck KGaA). Proteins from the nuclear fraction were extracted using a Nuclear Protein Extraction Kit (cat. R0050; Beijing Solarbio Science & Technology Co., Ltd.), according to the manufacturer's protocols. Protein concentrations were determined using a bicinchoninic acid assay protein quantification kit (Pierce; Thermo Fisher Scientific, Inc.). The protein samples were then heat-denatured, and 5 µg protein/lane was separated on a 10% SDS-PAGE gel, prior to transferral to polyvinylidene difluoride membranes. The membranes were subsequently blocked in 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA), which was prepared in a TBS-T buffer (50 mM Tris, pH 8.0, and 150 mM NaCl, containing 0.05% Tween-20) and incubated overnight at 4°C. Subsequently, the membranes were incubated with primary antibodies against anti-IRAK1 (cat. no. SC-5288; dilution, 1:400), anti-NF-κB p65 (cat. no. SC-8008; dilution, 1:400), anti-β-actin (cat. no. SC-47778; dilution, 1:1,000) (all from Santa Cruz Biotechnology, Inc.) and anti-histone H3 (cat. no. 4499; dilution, 1:2,000; Cell Signaling Technology, Inc.) at 4°C overnight. Following washing with TBS-T for 20 min, the membranes were incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies (cat. nos. 7076 and 7074; dilution 1:2,000; Cell Signaling Technology, Inc.) for 1 h at room temperature. The membranes were visualized using a Millipore Enhanced Chemiluminescence system (EMD Millipore; Merck KGaA) and analyzed using Image Lab (version 3.0; Bio-Rad Laboratories, Inc.). β-actin and histone H3 were used as internal controls. Relative protein expression, normalized to the internal control, is presented as a ratio of the blank control group.

Cells in the logarithmic growth phase were seeded into a 24-well plate with a density of 2×10⁵ cells/well. Subsequently, they were transfected with miR-146a mimic, mimic NC, miR-146a inhibitor or and inhibitor NC. For the blank control, the same amount of the Opti-MEM solution was used instead of RNA in the transfection. After 24 h of transfection, the THP-1cells were washed with Hank's balanced salt solution, and the culture medium was replaced with RPMI-1640 medium. LPS (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was added into culture medium at a final concentration of 1 µg/ml. Following treatment with LPS for 12 h, the culture medium was collected to measure IL-6 and TNF-α levels using ELISA kits (D6050 and DTA00D, R&D Systems, Inc.), according to manufacturer's protocols.

Target Scan bioinformatics software (version 7.2; www.targetscan.org) was used to predict the potential targets of miR-146a by typing the term ‘miR-146-5p’ into the search engine. The software revealed two conserved sites in the 3′-untranslated region (3′-UTR) of IRAK1. A wild-type IRAK1 mRNA 3′-UTR sequence containing the miR-146a target site was cloned into the XbaI site downstream of the firefly luciferase gene in the pGL-3-promoter vector (Promega Corporation) to obtain the wt-IRAK1-3′-UTR vector. In a parallel experiment, the IRAK1 mRNA 3′-UTR sequence was replaced by a mutant sequence (synthesized by ObiO Technology Co., Ltd.) to obtain the mut-IRAK1-3′-UTR vector. The THP-1 cells were seeded into 96-well plates (4×10⁴ cells/well) and cultured for 24 h. Subsequently, the cells were co-transfected with 1 µg plasmids and 100 nM miR-146a mimic using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. An empty pGL-3-promoter vector was used as the blank control, whilst an NC mimic was used as the negative control. The cells were subsequently lysed using Glo Lysis Buffer (Promega Corporation) at 24 h following transfection, and luciferase activity was measured using a Dual-Luciferase Reporter Assay system (Promega Corporation), according to the manufacturer's protocol. All luciferase activities were normalized to that of Renilla luciferase activity.

The data are presented as the mean ± standard deviation. One-way analysis of variance followed by the least significance difference post hoc test was performed to determine statistical significance among different groups using the SPSS software package (version 16.0; SPSS Inc.). P<0.05 was considered to indicate a statistically significant difference.

miR-146a mimicor miR-146a inhibitor were first transfected into THP-1 cells prior to RT-qPCR analysis. Transfection with miR-146a mimic significantly increased miR-146a expression (>400-fold) whilst transfection with miR-146a inhibitor significantly reduced miR-146a expression (by ~70%); with differences observed compared with the blank control or corresponding NC groups (P<0.01; Fig. 1A). IRAK1 mRNA expression was subsequently measured in THP-1 cells transfected with miR-146a mimic or inhibitor. miR-146a overexpression using the miR-146a mimic led to a significant reduction in IRAK1 mRNA expression (by ~88% relative to blank control; P<0.01 vs. mimic NC), whereas transfection with the miR-146a inhibitor led to a significant increase in IRAK1 expression (by ~20% relative to blank control; P<0.01 vs. inhibitor NC; Fig. 1B). At the protein level, cells transfected with the miR-146a mimic exhibited a significant reduction in IRAK1 expression compared with the mimic NC, whereas miR-146a inhibitor transfection significantly increased IRAK protein levels (P<0.01 vs. inhibitor NC; Fig. 2A and B). There were no significant differences between the blank control and to two corresponding NC groups (P>0.05 blank control vs. mimic NC and P>0.05 blank control vs. inhibitor NC; Fig. 2A and B).

THP-1 cells were treated with LPS to induce inflammation, which significantly augmented IRAK1 expression when compared with the blank control group (P<0.01; Fig. 3A and B). Under inflammatory conditions, transfection with miR-146a mimic significantly reduced IRAK1 expression, whereas transfection with miR-146a inhibitor significantly increased IRAK1 levels compared with the corresponding NC groups (P<0.01; Fig. 3B). As NF-κB is a downstream molecule of IRAK1 (17), western blot analysis was subsequently performed to measure the nuclear protein levels of p65; a subunit of NF-κB. Nuclear p65 levels were significantly higher in the LPS group compared with those in the blank control group (P<0.01; Fig. 3C). The changes in nuclear NF-κB p65 protein levels in cells transfected with either miR-146a mimic or miR-146a inhibitor mirrored those of IRAK1 levels in the corresponding treatment groups (Fig. 3B and C). These results suggested that miR-146a regulates NF-κB activation in the presence of LPS, by modulating the expression of IRAK1.

ELISA results demonstrated significantly increased IL-6 and TNF-α levels in the supernatant of cells in the LPS group when compared with cells in the blank control group (P<0.01; Fig. 4). miR-146a overexpression significantly reduced IL-6 and TNF-α secretion compared with the NC mimic groups (P<0.01). By contrast, transfection with the miR-146a inhibitor significantly increased IL-6 and TNF-α secretion when compared with the inhibitor NC group (P<0.01; Fig. 4). These findings suggested that LPS stimulation induced inflammatory responses in THP-1 cells by increasing the secretion of inflammatory cytokines; a process that is negatively regulated by miR-146a.

To investigate whether miR-146a can interact with the IRAK1 mRNA 3′-UTR, a luciferase reporter assay was performed. Prior to this assay, the predicted targets of this miRNA were revealed using TargetScan (version 7.2). Two evolutionarily conserved potential miR-146a target sites were discovered in the 3′-UTR of IRAK1 mRNA (positions 40–47 and 56–63; Fig. 5A). In the group co-transfected with miR-146a mimics and the wild-type 3′-UTR IRAK1 sequence, luciferase activity was significantly repressed when compared with the blank control group, while no significant difference was observed in cells co-transfected with the NC mimic (P<0.01; Fig. 5B). When the IRAK1 3′-UTR sequence was replaced with a mutant sequence (Fig. 5C), no significant differences were observed in the luciferase activity of cells co-transfected with the miR-146a mimic when compared with the blank control group (Fig. 5D). These results suggested that miR-146a directly targets the IRAK1 3′-UTR.