A total of 20 children aged 8-15 years with pneumonia (10 females and 10 males) and 20 gender and age-matched healthy children with fever were enrolled in the study. The 40 patients were recruited from the emergency department of Zhengzhou Children’s Hospital and they did not have any treatment before blood collection. We collected 5ml of peripheral venous blood from patients and obtained the serum via centrifugation. Parents of all the eligible patients accepted to participate in the study and gave informed written consent. The research process was reviewed and approved by Zhengzhou Children’s Hospital and the ethic committee of Zhengzhou Children’s Hospital.
Cell culture and cell transfection
The human WI-38 fibroblasts were obtained from American Type Culture Collection (ATCC, Manassas, USA). WI-38 cells were cultured in DMEM medium supplement with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin and maintained in a humidified incubator with 5% CO2 atmosphere at 37°C. LPS (5 μg/mL) was then added into WI-38 cells for 12h to induce inflammation.
MiR-146b mimic or inhibitor was applied for increasing or decreasing miR-146b expression in WI-38 cells. MyD88 vector was used for overexpression of MyD88. The mimic/inhibitor, vector and their negative control were all purchased from GenePharma Co. (Shanghai, China). The transfection was performed for 48h with the help of Lipofectamine 3000 reagent (Invitrogen, Carlsbad, USA) according to the manufacturer's introduction.
Quantitative real time-PCR (QRT-PCR)
Total RNA was extracted using miRNeasy Mini Kit (Qiagen, Valencia, USA). Multiscribe RT kit and TaqMan MicroRNA Assay (Applied Biosystems, Foster, USA) were applied for performing reverse transcription and quantifying qRT-PCR, respectively. U6 and GAPDH were carried out for normalizing miR-146b expression and MyD88 mRNA expression, respectively. The follows were the primers sequences: miR-146b forward primer: 5’-TGACCCATCCTGGGCCTCAA-3’, reverse primer: 5’-CCAGTGGGCAAGATGTGGGCC-3’; U6 forward primer: 5’-CTCGCTTCGGCA GCACA-3’, reverse primer: 5’-AACGCTTCACGAATTTGCGT-3’; MyD88 forward primer: 5′-CTCCTCCACATCCTCCCTTC-3′, reverse primer: 5′-GCTTGTGTCTCC AGTTGCC-3′; GAPDH forward primer: 5’-CTCGCTT CGGCAGCACA-3’, reverse primer: 5’-AACGCTTCACGAATTTGCGT-3’. All the data were analyzed through 2−ΔΔCT method.
Proteins were extracted from cells using by RIPA buffer (Beyotime Biotechnology, Beijing, China). BCA kit was used for determining protein concentration. An equal amount of protein was resolved on SDS-PAGE, followed by transferred to PVDF membranes (Bio-Rad, Hercules, USA). After blocking with 5% skim milk for 1h at 37℃, the membranes were incubated with primary antibodies overnight at 4 ℃, subsequently, the secondary antibodies for 1h at 37 ℃. Finally, the enhanced chemiluminescence reagent was carried out for monitoring the bands. For ensuring equal protein loading, GAPDH was served as the internal control. ImageJ 1.49 (National Institute of Health, Bethesda, MD) was used to quantify the intensity of the bands.
Cell Counting Kit-8 (CCK-8) assay
CCK-8 assay was applied for detecting cell viability. The cells were cultured in 96-well plates for 24h. Then, CCK-8 solution (10 μL) was added into each well and incubated for another 2h. Finally, the absorbance at 450 nm was detected by microplate reader (Bio-Tek, Winooski, USA). Cell viability was evaluated by the absorbance.
Enzyme-linked immunosorbent assay (ELISA)
The inflammatory cytokines in supernatant serum samples of children with pneumonia and in cell culture supernatant treated with LPS were quantified by corresponding ELISA kits (R&D Systems, Inc., Minneapolis, USA) following the manufacturer's protocols, including interleukin (IL)‑6, tumor necrosis factor (TNF)‑α and interleukin (IL)‑1β ELISA Kits. Then, a microplate reader was applied for measuring the absorbance at 450 nm. The concentrations of IL‑6, TNF‑α and IL‑1β were normalized and calculated based on the linear calibration curves obtained by standard solutions.
Luciferase reporter assay
The fragments of MyD88 3’UTR containing the wild-type (WT) or mutated binding sites of miR-146b were inserted into psiCHECK-2 vector (Promega, Madison, WI). Then, cells were co-transfected with luciferase reporter vectors and miR-146b mimic with the aid of Lipofectamine 2000. After transfection for 24h, the luciferase activities were analyzed by a luciferase reporter assay system.
All experiments were repeated in triplicates. The values were represented as mean ± SD and GraphPad Prism 6.0 software was applied for performing statistical analyses. The one-way Analysis of Variance (ANOVA) or student t-test was used to calculate P values between different groups. P value of <0.05 was considered as statistically significant.