Research subjects
Research subjects of this study included both pneumonia patients (n=62, 37 males and 25 females, 1 to 3 years, 1.7 ±0.5 years) and healthy controls (n=62, 37 males and 25 females, 1 to 3 years, 1.7 ±1.4 years). All patients and controls signed informed consent. These participants were enrolled at Inner Mongolia University for Nationalities Hulunbuir People's Hospital between March 2018 and March 2020. This study obtained ethics approval from aforementioned hospital Ethics Committee. The pneumonia patients were caused by either viral (n=34) or bacterial (n=28) infections. The patients excluded other diseases and initiated therapy. All the 62 healthy controls received a systemic-level physiological examination at aforementioned hospital during the same time period. Please check Table 1 for clinical data of patients and controls. Informed consent was obtained from all individual participants included in the study. All patients provided written form informed consent. Procedures operated in this research were completed in keeping with the standards set out in the Announcement of Helsinki and laboratory guidelines of research in China.
Preparation of plasma samples
All patients (before the initiation of therapy) and healthy controls were fasted overnight, followed by extraction of blood (5ml) from each participant. Centrifuged in EDTA tubes (1200g for 10 min at room temperature) was performed to separate the supernatant (plasma). RNA extractions were performed within 6h after plasma preparations
Human Bronchial Epithelial Cells (HBEpC)
HBEpCs (PromoCell) were used. Cells were cultivated in Bronchial Epithelial Cell Medium (ScienCell). Cells were collected from passage 4-6 were used for subsequent experiments. Cells were cultivated in medium containing 1, 5, and 10ng LPS for 48h in cases the treatment with LPS.
Vectors, miRNAs and transfections
Backbone vector expressing pcDNA3.1-GAS5 was constructed. MiR-155 mimic and negative control (NC) miRNA were purchased from Sangon (Shanghai, China). Expression vector (1μg) or miRNA (50 nM) was transfected into HBEpCs using lipofectamine 2000 (Invitrogen). Empty vector or NC miRNA transfections were performed to serve as NC groups. Untransfected cells were used as control (C) cells. Cells were cultivated for further 48h after transfections before subsequent experiments.
RNA samples
Plasma samples and HBEpCs were subjected to total RNA extractions using Ribozol (Invitrogen). DNase I (Sangon) incubation for 2h at 37 °C was perform to remove DNA. RNA integrity was checked using 5% Urine-PAGE gel.
Real-time quantitative PCR (RT-qPCR)
QuantiTect Reverse Transcription Kit (QIAGEN) was used to synthesize cDNA. After that, qPCRs were performed with cDNA samples as template using SYBR Green Master Mix (Bio-Rad) to measure the levels of GAS5 expression with GAPDH as internal control. To determine the level of mature miR-155 expression, poly (A) addition, reverse transcriptions and qPCRs were sequentially performed using GeneCopoeia All-in-One™ miRNA qRT-PCR Reagent Kit. The method of 2−ΔΔcq was used for Ct value normalizations.
Methylation analysis
DNeasy Tissue Kit (Qiagen) was used to extract genomic DNAs following manufacturer’s instructions. EZ DNA Methylation Lighting Kit was used to convert DNA. Routine PCR and MSP were performed and PCR products were separated using 2% agarose gel electrophoresis. Ultraviolet irradiation was used to visualize bands.
Apoptosis assay
Cells were cultivated in medium containing 10ng/LPS for 48h. After that, incubation with 70% ethanol was performed to achieve fixation. Following that, PI and Annexin-V FITC incubation was performed. Finally, apoptotic cells were analyzed by FACSCalibur instrument.
Statistical analysis
Three independent replicates were included in each experiment. Mean±SD values were used to express all data. Unpaired t test was used to compare patient and control groups. Multiple cell transfection groups were compared by ANOVA Tukey’s test. P<0.05 was statistically significant.