The cell lines (16HBECs and A549 cells) were purchased from the Type Culture Collection of the Chinese Academy of Science (Shanghai, China) and grown in Dulbecco's modified Eagle's medium (DMEM; Gibco^®; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal bovine serum (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and 100 µg/ml streptomycin (Beyotime Institute of Biotechnology, Shanghai, China) at 37°C in 5% CO₂.

An MTT assay (Beyotime Institute of Biotechnology, Shanghai, China) was used to measure cell viability. The cells were seeded in 96-well plates with 1–3×10⁴ cells in a volume of 200 µl in each well and incubated for 24 h. The cells were then incubated with different concentrations (0, 0.1, 1, 10 and 100 µg/ml) of lipopolysaccharide (LPS; Sigma-Aldrich; Merck KgaA) for 12 h at 37°C. The supernatant was removed from the cells and added to medium with 5 mg/ml MTT for 4 h. The medium was discarded and 100 µl solubilization solution (included in MTT assay kit) was added to dissolve the formazan crystals. The absorbance was measured at a wavelength of 570 nm using a Multiskan Spectrum instrument (Thermo Fisher Scientific, Inc.). The ratio of the absorbance of the treatment group to that of the control group represented the viability of the cells.

Cells seeded in 6-well plates at a density of 1×10⁶ cells/well were transfected with an miR-625-5p mimic (5′-AGGGGGAAAGUUCUAUAGUCC-3′), miR-625-5p inhibitor (5′-AGGGGGAAAGUUCUAUAGUCC-3′), a negative control miR (5′-UUCUCCGAACGUGUCACGU-3′) and an inhibitor negative control (5′-AAGAGGCUUGCACAGUGCA-3′), which were obtained from Guangzhou Ribobio Co., Ltd., (Guangzhou, China); and with the AKT2 small interfering RNA (si-AKT2; sense, 5′-GCUCCUUCAUUGGGUACAATT-3′), scrambled negative control (5′-UUCUCCGAACGUGUCACGUTT-3′), the AKT2 overexpression plasmid (v-AKT2), and control vector, which were obtained from Shanghai GenePharma Co., Ltd., (Shanghai, China). miR-625-5p mimic (75 pmol/well) or miR-625-5p inhibitors (120 pmol/well) and their controls (corresponding concentration) were mixed with 5 µl Lipofectamine 2000™ (Thermo Fisher Scientific, Inc.), then incubated with 1×10⁶ cells in 6-well plates. Similarly, si-AKT2 (75 pmol/well) or v-AKT2 (2.5 µg/well) were mixed with 5 µl Lipofectamine 2000™ and transfected into cells. Following 6 h, Opti-Minimum Essential Medium™ (Gibco; Thermo Fisher Scientific, Inc.) was discarded and fresh DMEM was added. The cells were incubated for another 24 h, then washed three times with cold PBS and used for protein extraction immediately after cell collection.

The cells seeded in 6-well plates at a density of 2×10⁵ cells/well were cultured with the miR-625-5p mimic or miR-625-5p inhibitor with LPS (1 µg/ml) for 24 h at 37°C. The cells and the culture supernatants were collected separately. The levels of interleukin (IL)-6 (cat. no. D6050) and tumor necrosis factor-α (TNF-α) (cat. no. DTA00D) in the supernatant released from 16HBECs were evaluated using ELISA kits (R&D Systems, Inc., Minneapolis, MN, USA).

Total RNA from cultured 16HBECs and isolated using RNAiso Plus (Takara Biotechnology Co., Ltd., Dalian, China). PrimeScript reagents (Takara Bio Inc., Otsu, Japan) were used to synthesize cDNA at 37°C for 15 min followed by RT inactivation at 85°C for 5 sec. RT-qPCR was completed using a MxPRO 3000 real-time PCR system and SYBR^® Premix Ex Taq™ (TliRNaseH Plus; cat. no. RR420) (Takara Bio, Inc.); 0.8 µl primers, 0.4 µl ROX Reference Dye or Dye II 2 µl cDNA and 8 µl dH₂O were employed and the reaction mixture was made up to 20 µl. The procedure was implemented according to the manufacturer's protocols. The thermocycling conditions were as follows: Denaturation at 95°C was for 5 sec, annealing at 60°C was for 34 sec min and extension at 60°C was for 1 min. The primers used are presented in Table I. The 2^−∆∆Cq relative quantification method was used to calculate the mean fold expression difference between the groups (15).

Proteins from 16HBECs were prepared routinely using radioimmunoprecipitation lysis buffer (cat no. P0013C) and phenylmethanesulfonylfluoride (cat no: ST506) kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. A bicinchoninic assay kit (Beyotime Institute of Biotechnology) was used to quantify the protein levels. Total protein extracts (40 µg) were separated by 10% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). Membranes were blocked in 5% bovine serum albumin (Beyotime Institute of Biotechnology; cat no. P0007) for 1 h at room temperature, then incubated with primary antibodies (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA). The primary antibodies were as follows: P-AKT2 (cat. no. 8599), AKT2 (cat. no. 2964), P-IκBα (cat. no. 9246), β-actin (cat. no. 4970) overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (1:3,000; cat. nos. 7076 and 7074; Cell Signaling Technology, Inc.) for 2 h at room temperature. The immunoreactive proteins on the blots were scanned by FluorChem FC3 (Protein Simple, San Jose, CA, USA).

The AKT2 3′UTR was amplified using human DNA as a template. The wild-type (WT) 3′UTR of AKT2 was inserted into the luciferase gene in the pMIR-Report vector (Promega Corporation, Madison, WI, USA; cat. no. E1330) and termed pMIR-AKT2-WT. The primers used to amplify the wild-type sequence were: 5′-CGAGCTCGGGAGGGGCCTGAAGAAGAACGA0-3′ forward, and 5′-CCAAGCTTCCTGGGCTTACTGGAGCTGCCA0-3′ reverse. pMIR-AKT2-MUT contained a mutated sequence in the 3′UTR of AKT2 inserted into the luciferase gene in the pMIR-Reporter vector. The primers used to amplify the mutant sequences were: 5′-AAGTTATATATGCGAAACCACCCAGCGGTGATGGCAGCGAG-3′ (mutated site underlined) forward; 5′-GTGGTTTCGCATATATAACTTTTTACTTAGCCTTTTTGGTT-3′ (mutated site underlined) reverse. All constructs were verified by direct sequencing by Sangon Biotech (Sangon Biotech Co. Ltd., Shanghai, China).

The biological software Microrna (http://www.mirbase.org/) and TargetScan release 7.2 (http://www.targetscan.org/) were used to predicted as a target gene of miR-625-5p. Cells (2×10⁴) were co-transfected in 96-well plates in triplicate with a firefly luciferase reporter plasmid (pMIR-AKT2-WT or pMIR-AKT2-MUT), a Renilla luciferase vector (pRL-SV40; Promega Corporation, Madison, WI, USA) and with miR-625-5p mimic or its control, using Lipofectamine 2000™. pRL-SV40 was used as a normalization control. Following 48 h, luciferase activity was determined using a Dual Luciferase Reporter Assay System (Promega Corporation, cat. no. E1910) according to the manufacturer's protocols and was expressed as the ratio of the firefly luciferase activity to the Renilla luciferase activity.

All experiments were repeated three times. GraphPad Prism version 5 (GraphPad Software, Inc., La Jolla, CA, USA) was used to analyze the data. The data are presented as the mean ± standard deviation for normally distributed data. A Student's t-test was used to assess the difference between two groups, while one-way analysis of variance was to analyze differences among three or more groups with a post hoc Student-Newman-Keuls test. P<0.05 was considered to indicate a statistically significance difference.

An MTT assay was performed to determine whether LPS influenced the viability of 16HBECs following 24 h of treatment with the various LPS concentrations (0, 0.1, 1, 10 and 100 µg/ml). LPS demonstrated a dose-dependent cytotoxic effect. A total of 1 µg/ml LPS was chosen as the best stimulatory concentration for further experiments (Fig. 1A). As presented in Fig. 1B, 16HBECs treated with LPS at 1 µg/ml significantly stimulated the cells' ability to secrete inflammatory cytokines compared with the control cells (IL-6 and TNF-α; P<0.05). 16HBECs expressed a significantly increased level of miR-625-5p compared with the A549 cell line following LPS induction (P<0.05; Fig. 1C); therefore, 16HBECs were selected for further analysis.

ELISA and RT-qPCR were used to confirm the association between miR-625-5p and inflammatory cytokine secretion. miR-625-5p expression was significantly increased in cells transfected with the miR-625-5p mimic (P<0.05; Fig. 2A). miR-625-5p had no effect on the production of IL-6 and TNF-α in control 16HBECs. However, the mRNA and protein levels of IL-6 and TNF-α were significantly decreased in miR-625-5p mimic transfected 16HBECs following LPS-induction (P<0.05; Fig. 2B-E). Transfection with the miR-625-5p inhibitor significantly decreased miR-625-5p expression in 16HBECs when compared with the inhibitor control following LPS induction (P<0.05; Fig. 3A). Transfection with the miR-625-5p inhibitor resulted in the significantly increased expression of inflammatory cytokine mRNAs (IL-6 and TNF-α) when compared with the control in LPS treated cells (P<0.05; Fig. 3B and C). As presented in Fig. 3D and E, inhibition of miR-625-5p expression significantly upregulated LPS-induced IL-6 and TNF-α secretion.

The AKT2 gene was predicted as a target gene of miR-625-5p using the biological software Microrna and TargetScan. A miR-625-5p binding site was identified in the 3′UTR of AKT2 (Fig. 4A). Overexpression of miR-625-5p significantly reduced the luciferase activity in cells transfected with the pMIR-AKT2-WT vector when compared with cells transfected with the pMIR-AKT2-MUT vector (P<0.05; Fig. 4B). Western blotting was then conducted to assess the effect of miR-625-5p on the AKT2 protein level. In the miR-625-5p mimic transfected cells, the protein level of AKT2 was significantly decreased when compared with the vector only cells (P<0.05; Fig. 4C).

AKT2 is involved in triggering the nuclear factor-κB (NF-κB) signaling pathway and induces inflammatory cytokines (16). To investigate whether miR-625-5p influenced the regulation of the phophoinositol-3-kinase (PI3K)/AKT pathway, the levels of phosphorylated AKT2 were quantified. The level of phosphorylated AKT2 pretreatment with the miR-625-5p mimic was significantly decreased compared with LPS treatment alone (P<0.05; Fig. 5A). Conversely, inhibition of miR-625-5p expression significantly increased the LPS-induced phosphorylation of AKT2 (P<0.05). The phosphorylation/degradation of inhibitor of κB (IκBα) is an essential step in the NF-κB signaling pathway (17). Phosphorylation of IκBα was assessed to determine whether the NF-κB signaling pathway is involved in the protective effect of miR-625-5p. The LPS-induced phosphorylation of IκBα was significantly suppressed by miR-625-5p overexpression and significantly increased by miR-625-5p inhibition compared with LPS treatment alone (P<0.05; Fig. 5B). To further confirm whether activation of the NF-κB pathway was dependent on the phosphorylation of AKT2, NF-κB activation (i.e., the phosphorylation of IκBα) was detected in LPS-stimulated 16HBECs pretreated for 24 h with si-AKT2 to knockdown AKT2 expression. Notably, treatment with si-AKT2 significantly inhibited the LPS-stimulated phosphorylation of IκBα (P<0.05), which demonstrated that the PI3K/AKT axis may be involved in NF-κB regulation in 16HBECs (Fig. 5C).