Microarray data
The four microarray expression profile datasets including GSE62306, GSE33336, GSE142237 and GSE117827 were obtained from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/). The dataset GSE62306, GSE33336 and GSE142237 are miRNA expression profiles. GSE62306 included nasal mucosa samples from 13 healthy children and 14 severely RSV-infected children. GSE33336 contained 11 patients with mild COPD and 20 patients with moderate COPD from lung tissue. GSE142237 had a total of 10 samples derived from bronchial epithelial cells, including 3 healthy controls and 7 asthmatics. GSE117827 was an mRNA expression profile containing 6 control healthy samples and 5 RSV-infected samples, derived from nasal mucosa samples of children after excluding other irrelevant samples.
Data preprocessing and differential miRNAs (DEmiRs) and gene (DEGs) screening
R software was used (version 4.0.3; https://www.r-project.org/) and bioconductor (http://www.bioconductor.org/) to process raw data. Sequencing platform files were used to convert probes and genes. When the genes had multiple probes, only the maximum probe corresponding data was taken. After the dataset was calibrated and normalized, the limma package was used for screening.
Prediction of target genes of miR-34b/c-5p and verification of target genes from DEGs
The online tool miRDB (http://mirdb.org/) was used to obtain the potential target genes of miR-34b/c-5p. The predicted target genes of the two miRNAs and the up-regulated mRNAs in the GSE117827 dataset were subjected to Venn analysis, and the significant up-regulated target genes were obtained after the intersection.
GO and KEGG pathway analysis
Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses (https://amp.pharm.mssm.edu/Enrichr/) were used to show functions and roles of the target genes.
Construction of PPI network and identification of the hub genes
The differentially expressed target genes were uploaded to the online tool, STRING database (https://string-db.org/). Then the Cytoscape software (version 3.7.1) was used to analysis the PPI networks basing on the STRING results. In order to find the modules of the whole network, the Molecular Complex Detection (MCODE) plug-in of the Cytoscape software was applied. The hub genes were identified by using the plug-in cytoHubba of the Cytoscape software, including Density of Maximum Neighborhood Component (DMNC) and Maximal Clique Centrality (MCC). The top ten genes with the highest connectivity were identified as key genes.
Cell culture and viral preparation
Human lung epithelial BEAS-2B cells and Hela cells were cultured in DMEM plus 10% fetal bovine serum (FBS) at 37°C and 5% CO2. RSV (Long strain/A2 type) was stored by the Department of Medical Microbiology, Central South University. RSV was propagated in Hela cells with DMEM containing 3% FBS and the viral titer was determined by plaque assay.
Transfection of miRNA mimics and RSV infection
Synthetic miR-34b/c-5p mimics (50 nmol/L) or negative control (RiboBIO, Guangzhou,China) were transfected into BEAS-2B cells following riboFECT CP Transfection Kit (RiboBIO, C10511-05, Guangzhou, China) for 24h. Subsequently, the cells were infected with RSV at MOI of 1 for 48h.
Dual-luciferase reporter assay
Simply, the 3′-UTR fragments of CXCL10 containing miR-34b/c binding site were amplified by Nanjing Genscript Biological Technology Co., Ltd. and cloned into the pmirGLO vector (Promega, USA). Then, the wild type of CXCL10 or mutant CXCL10 3′-UTR (CXCL10-WT or CXCL10-MUT) was constructed. Next, the constructed plasmids were co-transfected with miR-34b/c-5p mimics or mimic-NC into 293T cells by using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions. After 48 hours of transfection, luciferase activities were measured by the Dual-Luciferase Reporter System (Promega, USA).
Animal Models
6-8 weeks BALB/c female mice weighing 16–20 g (purchased from Hunan Tianqin Biological Technology Co., Ltd) were kept in a pathogen-free environment. The mice were randomly divided into the control group (n = 12) and the RSV group (n = 12). After anaestheting the mice with isoflurane, 5×106 pfu of RSV in 100 ul, was intranasally inoculated. For mock infections, mice were administered an equivalent volume of sterile PBS. The airway resistances of mice were tested on day 7 and 28 post-infection (6 mice from each group). Then the mice were sacrifice and their lungs were taken for follow-up studies. RSV infection was verified by indirect immunofluorescence (IFA) with RSV major surface glycoprotein G monoclonal antibody (Bioss,bs-1264R, Beijing, China) as a primary antibody and CY3-conjucted antibody (Boster, BA1032, Wuhan, China) as secondary antibody.
Real-time RT-PCR
Total RNA was extracted from lung tissue or cells using TRIzol reagent (Takara, Japan).Primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China),and the sequences were shown in Table 1.Each sample was reverse transcribed into cDNA using RR036A PrimeScript RT Master Mix (Perfect Real Time)(Takara, Japan).For miRNA cDNA synthesis, using RR037A PrimeScript RT Master Mix (Perfect Real Time)(Takara, Japan) with miRNA-specific stem-loop RT Primers. Then the cDNA was synthesized by reverse transcription and amplified using 2× SYBR Green qPCR Master Mix (Bimake, USA) according to the manufacturer’s instructions. qPCR was performed at 95°C for 3 min, and 40 cycles of 95°C for 15 s, 55°Cfor 30 s, and 72°C for 30 s. U6 was the internal reference for miR-34b/c, and GAPDH was the internal reference for the other target genes. The relative expression levels of mRNA were calculated using the 2−ΔΔCT method.
Measurement of airway responsiveness to methacholine
The airway resistances of mice were measured on the 7th and 28th day of RSV infection with by Buxco pulmonary function testing system (Buxco, Sharon, Connecticut, CT, USA). After anesthetizing the mice with sodium pentobarbital (60 mg/kg), the airway was separated and the tracheal intubation was performed, and then the mice were mechanically ventilated with a small animal ventilator at a tidal volume of 10 ml/kg, and a frequency of 120 breaths/min. Then the mice were inhaled 10μl of methacholine (0.00, 6.25, 12.5, 25, and 50 mg/mL) and the values of airway resistances (Rn,cm H2O s / mL) were obtained by measuring airway flow and pressure.
Hematoxylin-eosin staining
The left lung tissues taken from each animal were fixed in 10% formaldehyde solution for 24 h, and then embedded in paraffin and cut into 5 μm sections. Hematoxylin-eosin staining was performed according to routine experimental procedures, and then the histomorphological changes were observed under an optical microscope.
Immunofluorescence
The Lung tissue sections were fixed by 95% ethanol and 0.1% Triton-X100. The samples were then blocked with normal goat serum for 20 min, and incubated overnight with RSV major surface glycoprotein G monoclonal antibody (Bioss, bs-1264R, Beijing, China) rabbit primary antibody at 4 °C. Then the samples were incubated with a CY3-conjugated goat anti-rabbit secondary antibody (Boster, BA1032, Wuhan, China) at room temperature for 1 h. After counterstained with DAPI for 10 min, the samples were observed under a light microscope at high magnification (200×) (Leica, German).
ELISA
The levels of CXCL10 in the culture supernatants of BEAS-2B cells were measured using ELISA kits (Jianglaibio, Shanghai, China), according to the manufacturer’s protocol.
Statistical Analysis
GraphPad Prism software version 7.0 was used for statistical analysis, and the data were expressed as mean ± standard deviation. T-test was used for comparison between two groups; two-way ANOVA was used for comparison among multiple groups, and LSD was used for post hoc test. P < 0.05 was considered significant.