Newborn SD rats, (male; age, 3-8 days; weight, 10-15 g; Experimental Animal Center of Shandong University, Jinan, China) were injected with LPS intraperitoneally at 3 mg/kg to induce ALI 48 h after LPS administration. The rats were housed at room temperature (18-23˚C) with a 12 h-light/dark cycle. Rats were randomly fed with standard chow and water and adapted to experimental conditions at least 3 days before the experiment. All experiments were carried out following the ‘Guidelines for the Use of Laboratory Animal Care’ and approved by the Animal Care Use Committee of Shanxi Medical University (Taiyuan, China). All the rats (n=24) were randomly divided into a control and LPS group (n=12 each). The rats were anesthetized by administering intraperitoneal ketamine hydrochloride (60 mg/kg body weight) and xylazine hydrochloride (5 mg-kg body weight) (16-18). LPS (at 10 mg/kg body weight, E. coli O111:B4; Sigma-Aldrich; Merck KGaA) was dissolved in 20 µl sterile phosphate-buffered saline (PBS) for 24 h and then injected intratracheally. The control group received the same volume of PBS. The health and behaviors of rats were monitored every 12 h. Before the rats were sacrificed to collect the lung tissues, the rats were placed in a closed container with a low concentration of CO₂ (10-30%). Once the rats lost consciousness under the low concentration of CO₂ environment, they were euthanized with 100% CO₂ (the experiment was carried out in September 2019). The euthanasia was confirmed when lack of respiration and color fading of eyes were observed in the rats for a minimum of 1 min. All efforts were made to minimize their suffering. Preemptive euthanasia was performed for humane reasons if rats exhibited any of the following signs: Emaciated, gasping, no response to touch or an anal temperature <25˚C.

Clinical specimens were fixed with 10% formaldehyde at room temperature for 24 h and embedded in paraffin, then the sections (4 µm) were dewaxed and hydrated. Firstly, dewaxed sections were dried at 37˚C for 2 h, and its endogenous peroxide was blocked with 1% H₂O₂ for 5 min. Subsequently, the sections were washed 3 times with PBS, blocked with 5% BSA (cat. no. ST025; Beyotime Institute of Biotechnology) for 1 h at room temperature, and then incubated with anti-IRAK1 antibody (1:200; cat. no. ab238; Abcam) at 4˚C overnight. Subsequently, they were washed with PBS and incubated with goat anti-rabbit IgG H&L (HRP) secondary antibody (1:500; cat. no. ab205718; Abcam) for 1 h at room temperature. The sections were rinsed again and stained with 3,3-diaminobenzidine hydrochloride for 1 min. Finally, they were rinsed with double-distilled water and stained with hematoxylin and eosin (H&E) for 1 min at room temperature, and observed under a light microscope (magnification, x200).

The A549 cell line (cat. no. CCL-185™) was obtained from American Type Culture Collection (ATCC) and HPAEpiC (cat. no. 3200) (human type II alveolar epithelial cell) was purchased from ScienCell Research Laboratories, Inc. These two cell lines were incubated in Roswell Park Memorial Institute (RPMI-1640) medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum and 1% antibiotic antifungal solution (both from Sigma-Aldrich; Merck KGaA). In addition, the cells were cultured in an incubator with humidified CO₂ at 37˚C. miR-490-3p mimics (5'-CAACCUGGAGGACUCCAUGCUG-3') and its negative control (miR-NC, 5'-ACCGCUAAUCAUACGAAUACAC-3') were obtained from Shanghai GenePharma Co., Ltd. The cells (1x10⁵) were seeded in 12-well plates and transfected with miR-490-3p or miR-NC (50 pmoles) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37˚C for 24 h according to the manufacturer's instructions. Cells were used for subsequent experiments after 24 h. Four groups were designed, namely the control group, the LPS group, the LPS + NC group and the LPS + miR-490-3p. LPS (100 ng/ml) was used to treat the cells for 4 h.

Total RNA was extracted from the cells using TRIzol^® reagent (Thermo Fisher Scientific, Inc.) and reverse-transcribed into cDNA with a cDNA Synthesis Kit (Sangon Biotech Co., Ltd.) according to the manufacturer's protocol. RT-qPCR was conducted with a SYBR Prime Script RT-PCR kit (Invitrogen; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: 10 min at 95˚C; 40 cycles of 1 min at 95˚C, 2 min at 63˚C and 1 min at 72˚C. The primers were synthesized by Sangon Biotech Co., Ltd. The miRNA relative expression was calculated by normalizing to U6 small nuclear RNA. The primer sequences were as follows: miR-490-3p forward, 5'-CGTGGATCCTTCTTCA ACCAACGGTGGTG-3' and reverse, 5'-CCAGAATT CAAAGCAGGAAGAGTAAGACTTCC-3'; IRAK1 forward, 5'-CCTCCAGGTTCCACTCTCTG-3' and reverse, 5'-AACCACCCTCTCCAATCCTG-3'; TRAF6 forward, 5'-TTGCACATGAGACTGTTGGC-3' and reverse, 5'-CTTCGAATGGTCCGCTTGAG-3'; GAPDH forward, 5'-AACGGATTT GGTCGTATTG-3' and reverse, 5'-GGAA GATGGTGATGGGATT-3'; U6 forward, 5'-GGAGCGAG ATCCCTCCAAAAT-3' and reverse, 5'-GGCTGTTGTCAT ACTTCTCATGG-3'. Relative expression was calculated using the 2^-ΔΔCq method (19), using U6 or GAPDH as normalization controls.

RIPA lysate (Roche Diagnostics) was used to isolate the total proteins from both lung tissues and cells. Protein concentration was determined using a BCA kit (Beyotime Institute of Biotechnology). A total of 50 µg of protein was loaded on a 12% polyacrylamide gel and electrophoresed at 100 V for 2 h and electrically transferred to polyvinylidene fluoride (PVDF) membranes. After being blocked with 5% skimmed milk (room temperature; 1 h), the membranes were washed 3 times with TBS-0.1% Tween for 10 min each time, then incubated with antibodies of anti-IRAK1 (1:1,000; cat. no. ab238), anti-TRAF6 (dilution 1:1,000; cat. no. ab227560), anti-phosphorylated (p-)NF-κB (dilution 1:1,000; cat. no. ab76302) and anti-NF-κB (dilution 1:1,000; cat. no. ab32536; all from Abcam) at 4˚C overnight. Then, the membranes were washed with TBST again and incubated with goat anti-rabbit IgG H&L (HRP) (dilution 1:3,000; cat. no. ab205718; Abcam) at room temperature for 1 h. Subsequently, the membranes were washed 3 times, 10 min each with TBST. Finally, Pierce™ ECL Western Blotting Substrate (Invitrogen; Thermo Fisher Scientific, Inc.) was used for imaging, and ImageJ (v1.48; National Institutes of Health) was adopted to semi-quantify the gray value of each protein.

Cells of each group in the logarithmic growth phase were trypsinized, centrifuged (500 x g) for 5 min at room temperature and counted, and then inoculated in 96-well plates at 2x10³ cells/well. After 24 h of incubation (at 37˚C with 5% CO₂), the culture solution was removed. After treatment according to the experimental group, 10 µl of CCK-8 solution (Beyotime Institute of Biotechnology) was added to each well and incubated at 37˚C for 1 h. A microplate reader was used to detect the absorbance value of each well at 450 nm. Four parallel wells were set in each group, and each experiment was repeated 3 times.

The cells were seeded in a 12-well plate at 1x10⁵ cells/well and incubated at 37˚C with 5% CO₂ in a humidified environment for 12 h until fully adherent. After treating the cells of each group, BrdU at a final concentration of 30 μmol/l was added and incubated at 37˚C for 4 h. Subsequently, the cell slides were washed with PBS and fixed with 4% formaldehyde at room temperature for 10 min, and then acidified and denatured in PBST (PBS with 0.1% Tween-20) containing 2 mol/l of hydrochloric acid for 30 min. After being blocked with PBST containing 5% bovine serum albumin (Beyotime Institute of Biotechnology) for 1 h, the cells were incubated with BrdU antibody (1:1,000 in PBS; cat. no. ab8152; Abcam) for 2 h at room temperature. Then the cells were washed 3 times with PBS, and incubated with goat anti-mouse IgG H&L (Alexa Fluor^® 647; cat. no. ab150115; Abcam) (1:1,000 in PBS) for 1 h. Finally, DAPI (Beyotime Institute of Biotechnology) was used for labeling the nuclei, and BrdU was observed with a fluorescence microscope (magnification, x200). The cell proliferation rate was calculated as follows: Cell proliferation rate = Number of positive cells/number of total cells x100%. The experiment was repeated three times.

The supernatant of cell culture media was collected and centrifuged on a low-speed centrifuge (500 x g) for 5 min at room temperature. Then the expression of IL-6, TNFα and IL-1β in the supernatant was detected using ELISA kits for IL-6 (cat. no. 70-EK206/3-96), TNF-α (cat. no. 70-EK282/3-96) and IL-1β (cat. no. 70-EK201B/3-96) according to the manufacturer's instructions. All of the detection kits were obtained from Hangzhou Multi Sciences (Lianke) Biotech, Co., Ltd.

Bioinformatic analysis was conducted to predict the downstream target of miR-490-3p using StarBase v3.0 (http://starbase.sysu.edu.cn) (20). The amplified DNA sequence was cloned into pmirGLO Dual-luciferase vectors (Promega Corporation) to construct wild-type (WT) IRAK1 3'-untranslated region (UTR) and mutant (MUT) IRAK1 3'-UTR reporter vectors. A549 cells (1x10⁵) were seeded in 24-well plates overnight and then transfected with IRAK1-WT or IRAK1-MUT reporter vector and transfected with miR-490-3p mimics or negative controls using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). A Dual-luciferase reporter gene assay system (Promega Corporation) was adopted to determine luciferase viability 48 h after transfection. Renilla luciferase activity was used as the internal control.

SPSS (version 20.0; IBM Corp.) was used for data analysis. All data were expressed as the mean ± SD. Statistical analysis was performed using an unpaired Student's t-test. Differences between the two groups were analyzed using χ². The correlation of the molecules was analyzed using Pearson's correlation test. P<0.05 was considered to indicate a statistically significant difference.

To probe miR-490-3p function in ALI, an LPS-induced ALI rat neuronal model was established and the pathological alteration in lung tissue of LPS rats was examined using H&E staining (Fig. 1A). As a result, it was revealed that there was edema, lung tissue alteration, incomplete nuclear staining, unclear cell boundaries and accumulating inflammatory cell and neutrophil infiltration in the lungs of the LPS-treated rats (Fig. 1A). In addition, RT-qPCR revealed that miR-490-3p was downregulated in LPS-induced ALI (Fig. 1B). Furthermore, it was also revealed that the inflammatory factors IL-1β, IL-6 and TNFα were significantly upregulated in the lung tissues of LPS-induced ALI compared with the control group (Fig. 1C). Furthermore, Pearson correlation analysis revealed that miR-490-3p was negatively correlated to IL-1β, IL-6 and TNFα expression in LPS-induced ALI (Fig. 1D). The aforementioned findings indicated that miR-490-3p was downregulated in LPS-induced ALI, and inversely related to the inflammatory response.

The relative expression of IRAK1 and TRAF6 in various groups was examined by RT-qPCR. The results indicated that both IRAK1 and TRAF6 mRNAs were significantly upregulated in LPS-induced ALI (Fig. 2A). In addition, it was confirmed that miR-490-3p was negatively correlated with IRAK1 and TRAF6 expression (Fig. 2B). Moreover, immunohistochemistry indicated that the number of IRAK1-positive cells were increased in LPS-induced ALI (Fig. 2C). Furthermore, western blot analysis demonstrated that the expression of IRAK1, TRAF6 and p-NF-κB proteins in LPS-induced ALI was significantly enhanced compared with that of the control group (Fig. 2D and E). These studies indicated that IRAK1 and TRAF6 were upregulated in ALI and were inversely related to miR-490-3p.

miR-490-3p-overexpressed A549 cells were constructed to further investigate its mechanism in LPS-induced ALI (Fig. 3A). RT-qPCR revealed that miR-490-3p was increased in the LPS + miR-490-3p-treated group compared with the LPS + NC-treated group (Fig. 3B). Moreover, cell proliferation of LPS + miR-490-3p was enhanced compared with the LPS + NC-treated group (Fig. 3C). Furthermore, BrdU assays affirmed that the cell viability of the LPS + miR-490-3p group was significantly increased compared with the LPS + NC group (Fig. 3D). ELISA assays also indicated that the inflammatory factors IL-1β, IL-6 and TNFα were decreased in the LPS + miR-490-3p-treated group compared with the LPS + NC group (Fig. 3E). These studies indicated that miR-490-3p overexpression significantly inhibited LPS-induced cell injury and inflammatory response.

To further study the miR-490-3p mechanism in LPS-induced ALI, RT-qPCR and western blot analysis was used to examine the relative expression of IRAK1, TRAF6 and p-NF-κB. It was revealed that the mRNA and protein expression of IRAK1, TRAF6 and p-NF-κB were significantly downregulated in the LPS + miR-490-3p treatment group when compared with the LPS + NC treatment group (Fig. 4A-D). As a result, miR-490-3p appeared to inhibit the LPS-activated IRAK1/TRAF6/NF-κB pathway.

To identify the downstream molecular mechanism of miR-490-3p, its candidate targets were analyzed via StarBase (http://starbase.sysu.edu.cn). The results indicated that miR-490-3p could bind to IRAK1 (Fig. 5A). Similarly, the targeted binding relationship between miR-490-3p and IRAK1 was verified using a dual luciferase reporter assay. miR-490-3p mimics were revealed to attenuate the luciferase viability of IRAK1-WT but had no significant effects on that of the IRAK1-MUT (Fig. 5B). Thus, these results verified that miR-490-3p decreased LPS-induced inflammation by directly targeting IRAK1.