All experimental procedures in the present study were approved by the Experimental Animal Ethics Committee of Sichuan University (Chengdu, China). RT-qPCR for miRNA was performed using an EpiScript™ Reverse Transcriptase kit (EpiCentre, Palmerston North, New Zealand), as reported previously (21). A total of 20 male Sprague Dawley rats (age, 5–7 weeks; weight, 160–200 g; Dashuo Center of Experimental Animals, Chengdu, China), were used for the experiments of the present study. The animals were housed in standard conditions at 22±1°C with relative humidity of 59% and a 12 h light/dark cycle, and were provided with ad libitum access to food and water. The rats were randomly divided into two experimental groups: A control group (n=10) and a silicosis group (n=10). The rats were then sacrificed through loss of blood under 10% chloral hydrate anesthesia. Following sacrifice on day 40, the lungs were harvested for total RNA isolation and histological analysis. Briefly, 5 µm sections of lung tissue were obtained from the inferior lobe of the left lungs and fixed with 10% formalin (Kelon, Beijing, China), embedded in paraffin (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) and stained with hematoxylin and eosin (Beijing Solarbio Science & Technology Co., Ltd.) and Masson (Beijing Solarbio Science & Technology Co., Ltd.) dyes for histological examination of the collagen fibers. The tissue samples were cryopulverized using Biopulverizer™ (Biospec Products, Inc., Bartlesville, OK, USA) and homogenized using a Mini-Bead-Beater-16 (BioSpec, Shanghai Yuan Sheng Co., Ltd., Shanghai, China). Total RNA was isolated from the tissue samples using TRIzol^® (Invitrogen Life Technologies, Carlsbad, CA, USA) and further purified using an RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Total RNA (600 ng), 10 µM stem-loop RT primer (Table I), 10X RT buffer, 2.5 mM each of dATP, dGTP, dCTP and dTTP (HyTest Ltd., Turku, Finland), 10 U/µl reverse transcriptase and 40 U/µl RNase inhibitor (EpiCentre) were subjected to RT reactions. PCR Master Mix (Qiagen) and 0.4 µM of each primer (Table I) were used for RT-qPCR. The reactions were conducted with the following thermocycling conditions: 95°C for 5 min, followed by 40 cycles of 95°C for 10 sec, and 60°C for 60 sec. Each sample was normalized to endogenous U6 RNA content. RT-qPCR was performed using a CFX96 Touch™ Real-Time PCR Detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Each experiment was performed in triplicate. The Data were analyzed using Microsoft Excel. The relative expression levels of miR-146a were evaluated by the 2^−ΔΔCT method.

NR8383 cells were purchased from Shanghai Institute of Biochemistry and Cell Biology and were cultured in Ham's F12K medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 15% fetal bovine serum (Gibco Life Technologies, Grand Island, NY, USA). The NR8383 cells were seeded in triplicate into 6-well plates at a density of 1×10⁵ cells/well, and were grown to 30-50% confluence at 37°C in an atmosphere containing 5% CO₂. Turbofect transfection reagent from Thermo Fisher Scientific (Pittsburgh, PA, USA) was used for transfection, according to the manufacturer's instructions. The miRNA mimic control, miR-146a mimic, miR-181b mimic, miRNA inhibitor control, miR-146a inhibitor and miR-181b inhibitor (Guangzhou RiboBio Co. Ltd., Guangzhou, China) were transfected at a final concentration of 25 nM. Following 24 h of transfection, >90% of the cells exhibited green fluorescence under an inverted fluorescence microscope (Nikon ECLIPSE Ti-U; Nikon, Tokyo, Japan).. The cells were then treated with 20 µg/cm² SiO₂ (cat. no. S5631; Sigma-Aldrich). The silica content of the quartz dust was >99%, particle size of the dust was 0.5–10 µm, and 80% of the dust particles were 1–5 µm. The cells and culture supernatants were collected following SiO₂ treatment for 12 h at 37°C in an atmosphere containing 5% CO₂. RT-qPCR, western blotting and ELISA were then performed to detect the expression levels of TNF-α and IL-1β.

The NR8383 cells were suspended at ~1×10⁷ cells/well in TRIzol^® reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), and the total RNA was isolated, according to the manufacturer's instructions. Total RNA (1 µg) was subjected to reverse transcription using the PrimeScript™ RT reagent kit with gDNA Eraser (Takara Bio, Inc., Tokyo, Japan). SYBR^® Premix Ex Taq™ II (Takara Bio, Inc.) and the following specific primers were used for RT-qPCR: TNF-α, forward 5′-CAT GGA TCT CAA AGA CAA CCAA-3′ and reverse 5′-CTC CTG GTA TGA AAT GGC AAA T-3′; IL-1β, forward 5′-CTT CAA ATC TCA CAG CAG AAT C-3′ and reverse 5′-GCT GTC TAA TGG GAA CAT CAC A-3′; β-actin, forward 5′-GGA GAT TAC TGC CCT GGC TCC TA-3′ and reverse 5′-GAC TCA TCG TAC TCC TGC TTG CTG-3′. The PCR reactions were run using the following thermal cycling parameters: 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec, 60°C for 15 sec and 72°C for 45 sec. Each experiment was performed in triplicate.

Total protein (30 µg) was extracted using a total protein extraction kit (Nanjing KeyGEN Biotech Co., Ltd., Nanjing, China) according to the manufacturer's instructions, and loaded onto 15% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA) using a semi-dry blotting system (Invitrogen Life Technologies). The membranes were blocked with 5% skim milk (w/v) containing 0.2% Tween-20 (Sigma-Aldrich) for 2 h at room temperature, and then were incubated overnight with the following primary antibodies in blocking buffer at 4°C: Polyclonal goat anti-rat TNF-α (1:300; cat. no. sc-1349; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and polyclonal goat anti-rat IL-1β (1:500; cat. no. sc-1251; Santa Cruz Biotechnology, Inc.). These were used according to the manufacturer's instructions. The membranes were washed three times for 6 min with 1X Tris-buffered saline with Tween-20 were then incubated with polyclonal rabbit anti-goat secondary antibody (1:2,000; cat no. ZB-2306; ZSGB-BIO, Beijing, China) for 1 h at 37°C in an atmosphere containing 5% CO₂. Following final washes, the signals were detected using enhanced chemiluminescence reagents (Beyotime Institute of Biotechnology, Shanghai, China). The intensity of each signal spot was transformed into digital data, with auto-background subtraction during spot density analysis, using the Image-Pro Plus software, version 6.0 (Media Cybernetics, Inc., Bethesda, MD, USA).

The levels of TNF-α and IL-1β in the supernatants were determined using TNF-α and IL-1β ELISA kits (NeoBioscience Technology Co., Ltd., Beijing, China), according to the manufacturer's instructions. Briefly, the samples were diluted and added to the wells (100 µl) prior to being covered with a closure plate membrane, and incubated for 90 min at 37°C. The closure plate membrane was removed and the liquid was carefully discarded, prior to five washes in phosphate-buffered saline containing 1% bovine serum albumin and 0.05% Tween 20, each for 30 sec. Horseradish peroxidase-conjugated anti-rat TNF-α or IL-1β polyclonal antibody (from the TNF-α and IL-1β ELISA kits; NeoBioscience Technology Co., Ltd.; 100 µl) was added to each well, except the blank well, and incubated for 60 min at 37°C. The closure plate membrane was removed, the liquid discarded, and the plates were dried prior to further washing with washing buffer, repeated five times for 30 sec. A total of 100 µl Chromogen Solution B (NeoBioscience Technology Co., Ltd.) was added to each well, and incubated in the dark for 15 min at 37°C. A total of 100 µl stopping solution (NeoBioscience Technology Co., Ltd.) was then added to each well to stop the reaction. Absorbance was measured at 450 nm using a Thermo Scientific Multiskan GO (Thermo Fisher Scientific, Pittsburgh, PA, USA) as compared with the blank well 15 min following the end of the reaction.

R software from the The Comprehensive R Archive Network (http://cran.r-project.org/) was used to perform all statistical analyses. A one-way analysis of variance and two-tailed Student's t-test were used to analyze the data. P<0.05 (two-tailed) was considered to indicate a statistically significant difference.

Typical fibrosis was observed in the SiO₂-treated lungs, which involved infiltration of macrophages in alveolar spaces and increased collagen deposition (21). miRNA array analysis of the total RNA samples was performed on the day 40-SiO₂-treated lungs, and these were compared with the respective controls, as described in our previous study (21). Of 1,890 miRNAs examined, 39 miRNAs were either up- or downregulated (P<0.05; fold changes >2) in the SiO₂-treated lungs (21). In addition, the results of the RT-qPCR revealed that the expression of miR-146a was significantly increased and the expression of miR-181b was significantly decreased in the SiO₂-treated lungs (Fig. 1).

As shown in Fig. 2A, when the NR8383 cells were transfected with the miR-181b mimic for 24 h following 12 h of SiO₂ treatment, the expression of TNF-α was significantly decreased, compared with the miRNA mimic control group (P<0.05). Simultaneously, miR-181b inhibitors increased the mRNA expression of TNF-α significantly (P<0.05; Fig. 2B), whereas the miR-146a mimic and inhibitors had no significant effect on the mRNA expression levels of TNF-α (Fig. 2A and B). As shown in Fig. 2C, 12 h following SiO₂ treatment of the NR8383 cells transfected with either the miR-146a mimics, miR-181b mimics or miRNA mimic control, the miR-146a mimics significantly attenuated the expression of IL-1β (P<0.05). The expression levels of IL-1β in response to 12 h of SiO₂ treatment, subsequent to transfection with the miR-146a or miR-181b inhibitors were significantly increased, compared with those in the inhibitor control group (P<0.05; Fig. 2D).

The results of the western blot analysis demonstrated that overexpression of miR-181b following SiO₂ treatment for 12 h significantly reduced the protein levels of TNF-α, compared with the miRNA mimic control group (P<0.05; Fig. 3A). Subsequent to transfection with miR-181b inhibitors, followed by SiO₂ treatment for 12 h, the protein levels of TNF-α were significantly upregulated (P<0.05; Fig. 3B). However, the miR-146a mimics or inhibitors had no significant effect on the protein expression levels of TNF-α. The NR8383 cells transfected with the miR-146a mimic exhibited lower expression levels of IL-1β in response to the subsequent SiO₂ stimulation (P<0.05; Fig. 3C) and IL-1β was significantly upregulated in the NR8383 cells, which were transfected with the miR-146a or miR-181b inhibitors (P<0.05; Fig. 3D).

When the NR8383 cells were transfected with the miR-181b mimic, the expression of TNF-α was significantly decreased in the culture supernatants, compared with the miR-181b mimic control group (P<0.05; Fig. 4A). Simultaneously, miR-181b inhibitors increased the protein levels of TNF-α significantly (P<0.05), while the miR-146a mimic and inhibitors had no effect on the protein expression of TNF-α (Fig. 4A and B). As shown in Fig. 4C, when the NR8383 cells were transfected with miR-146a or miR-181b mimics for 24 h, followed by SiO₂ treatment for 12 h, the expression levels of IL-1β were significantly decreased, compared with the mimic control group (P<0.05). The expression of IL-1β in response to SiO₂ treatment, following transfection with miR-146a or miR-181b inhibitors, was significantly increased, compared with the inhibitor control group (P<0.05; Fig. 4D).