2.1 Specimen collection
Twenty-six children each with asthma and in good health, respectively, in Shengjing Hospital of China Medical University (Shenyang, China) from August 2016 to February 2017 were included. For the diagnostic criteria for asthma, we referred to the Guidelines for the Diagnosis and Prevention of Bronchial Asthma in Children revised by the Respiratory Group of Pediatrics Society of Chinese Medical Association in 2016. Exclusion criteria for childhood asthma included an acute respiratory infection or glucocorticoid treatment within 4 weeks before the hospital visit. Inclusion criteria for the healthy control group were as follows: 1. No definite abnormality found in routine physical examination indicators in the laboratory 2. No history of underlying diseases or drug therapy before blood collection; 3. Matching mean age and sex composition with asthma group. Approximately 5 ml of venous blood was collected from all subjects, along with clinical data and laboratory test results. This study was approved by the ethics committee of Shengjing hospital, China Medical University, and all patients signed written informed consent. The clinical information of the patients is presented in Table 1.
Table 1
Basic information about the subjects.
|
|
Chip analysis group
|
qRT-PCR test group
|
Asthm (n=3)
|
control (n=3)
|
Asthm (n= 26)
|
control (n=26)
|
Age (year)
|
8.00±1.76
|
7.80±1.28
|
7.53±2.16
|
6.83±1.83
|
gender
|
2/1
|
2/1
|
17/9
|
18/8
|
FEV1/FVC (%)
|
66.39±10.34*
|
84.21±7.03
|
68.09±13.68**
|
86.99±6.54
|
FEV1 (L)
|
2.48±0.83*
|
3.42±0.58
|
2.31±0.73**
|
3.32±0.48
|
FEV1 (%)
|
77.36±14.24*
|
101.45±10.42
|
80.36±6.49**
|
100.40±1.12
|
FVC (L)
|
3.52±0.69*
|
3.90±0.72
|
3.38±0.87**
|
3.83±0.56
|
FVC (%)
|
0.90±0.21
|
1.17±0.17
|
1.03±0.18
|
1.08±0.15
|
PEF (L)
|
5.52±2.10*
|
7.91±1.83
|
5.36±1.91**
|
8.97±2.37
|
FeNO (ppb)
|
47.47±20.07*
|
13.28±8.74
|
60.42±12.56**
|
15.33±7.24
|
IgE CIU/mL)
|
187.00±98.80*
|
58.32±6.43
|
198.33±104.23**
|
56.52±9.39
|
Note: Values are presented as means ± SD unless otherwise stated. Abbreviations: FEV1, forced expiratory volume in the first second; FVC, forced vital capacity; PEF, peak expiratory flow; FeNO, fractional exhaled nitric oxide; IgE, immunoglobulin E; SD, standard deviation. *p<0.05, ** *p<0.01. |
2.2 Arraystar human lncRNA/mRNA expression profile microarray analysis
Peripheral blood mononuclear cell samples (PBMCs) were isolated by Ficoll density gradient centrifugation within 3 h of collecting the 5 ml-venous blood samples. Six PBMC samples (3 asthma patients and 3 healthy control patients) were selected and analyzed by lncRNA/mRNA expression microarray. The chip used Arraystar Human LncRNA Microarray V4.0 (Arraystar, Rockville, MD, USA), and the detection and data processing of the gene chip were assisted by Kangcheng Biological Engineering Co. Ltd. (Shanghai, China). The GeneSpring GX software V12.1 software (Agilent Technologies, Palo Alto, CA, USA) was used for quantile standardization of the original data.
2.3 Differential expression analysis and gene enrichment analysis
“Limma” package was used to select the DE lncRNAs and mRNAs between patients with asthma and healthy patients (control) based on the screening criteria of p<0.05 and log∣FC∣>2. Then, the DE mRNAs were imported into the DAVID 6.8 database (https:/ /david.ncifcrf.gov/) for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Thus, the functional level and biological pathway analyses results of genes were obtained. A p<0.05 indicated statistical significance.
2.4 Protein-protein interaction network analysis
The DE mRNAs were imported into the String database (https://string-db.org/) to obtain the interaction between proteins expressed by genes. Subsequently, we visualized this interaction and selected the five genes with the highest connectivity as hub genes.
2.5 Construction of the ceRNA network
Pearson correlation analysis was used to obtain the correlation coefficient between DE lncRNAs and hub genes. p<0.05 and r>0.9 were selected as screening criteria. Hub gene-related lncRNAs were screened out, and the target miRNAs of the lncRNAs were searched through the lncRNA Base database (http://starbase.sysu.edu.cn/starbase2/mirLncRNA.php). The related gene pairs between hub mRNAs and miRNAs were searched through the miRWalk database (http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/). Then, the miRNA-mRNA pairs and the lncRNA-miRNA pairs were intersected to obtain the lncRNAs-miRNA-mRNA gene pairs.
2.6 Cell culture and vectors transfection
The human bronchial epithelial cell line (16HBE cells) was purchased from the cell bank of Xiangya Medical College, Central South University (Hunan, China). The 16HBE cells were inoculated into a complete medium (DMEM high sugar medium, containing 10% fetal bovine serum, 1% NEAA, 1% L-glutamine, 1% sodium pyruvate, and 100 μg/ml penicillin-streptomycin mixture). Next, the 16HBE cells were cultivated in a 5% CO2 incubator and fully saturated humidity at a constant temperature of 37 ℃. The downregulation vectors for lncRNA SNHG15, miR-192-3p mimics, and inhibitors were designed and synthesized by GenePharma Co., Ltd. (Suzhou, China). Lipofectamine 3000 was purchased from Invitrogen (MA, USA).
2.7 Real-time qPCR
We extracted total RNA from PBMCs and 16HBE cells using TriZol Reagent (Invitrogen, USA). cDNA was synthesized using All-in-One™ First-Strand cDNA Synthesis Kit FulenGen, Guangzhou, China) and amplified using SYBR green (Applied Biosystems LLC, Bedford, MA, USA). Then, the 2-ΔΔCt method was performed to calculate the relative gene expression with β-actin and U6 serving as the internal references. The RT-qPCR primer sequences are presented in Table 2.
Table 2
RT-qPCR primer sequences.
Gene
|
Sequences of the primers
|
Annealing temperature (℃)
|
Product length (bp)
|
β-actin
|
F:5' GTGGCCGAGGACTTTGATTG3'
R:5’ CCTGTAACAACGCATCTCATATT3’
|
60
|
73
|
RP11-598F7.3
|
F:5' GAAACTGTTATTCAGGAGACAACTA 3’
R:5’ TATTTCTTGTGCTTTGTTTTATTTT 3’
|
60
|
174
|
NR_002734
(PTTG3P)
|
F:5' TCTGGTTGAGAGCGGCAATAA 3’
R:5’ AGGATGCCTGGTTCTTCGTT 3’
|
60
|
77
|
U6
|
F:5’GCTTCGGCAGCACATATACTAAAAT3’
R:5’CGCTTCACGAATTTGCGTGTCAT3’
|
60
|
89
|
hsa-miR-192-3p
|
GSP:5’GGCTGCCAATTCCATAGG3’
R:5'GTGCGTGTCGTGGAGTCG3’
|
60
|
62
|
hsa-miR-6835-3p
|
GSP:5’GGGCAAAGCACTTTTCTGTC3’
R:5'GTGCGTGTCGTGGAGTCG3’
|
60
|
64
|
β-actin
|
F:5' GTGGCCGAGGACTTTGATTG3'
R:5’ CCTGTAACAACGCATCTCATATT3’
|
60
|
73
|
CCNA2
|
F:5' AGTGGAGTTGTGCTGGCTAC3’
R:5’ CACAGTCAGGGAGTGCTTTC3’
|
60
|
223
|
CCNB1
|
F:5' TGAGCCTATTTTGGTTGATACTGC3’
R:5’ GCAAGAATTACATCAGAGAAAGCCT3’
|
60
|
105
|
2.8 Western blot
RIPA lysis buffer (Beyotime, Shanghai, China) was used to extract the total protein. Protein concentration was measured with BCA Protein Quantitative Kit (Kangrun Biology Co., Ltd., Guangzhou, China). A 10% SDS-PAGE gel was used to separate 40 µg proteins, which was then transferred onto a polyvinylidene fluoride (PVDF) membrane (Kangrun Biology, Guangzhou, China). Subsequently, the membranes were blocked with 5% milk for 1.5 h at room temperature. Subsequently, the primary antibodies against GAPDH (1:10,000; Abcam, Cambridge, UK), E-cadherin (1:1000; Abcam), α-SMA (1:1000; Abcam), and vimentin (1:1000, Abcam) were added to the membrane for coculturing overnight at 4 °C. Then, the membranes were cocultured with the secondary antibody for 2 h at room temperature. Finally, the membranes were exposed and developed with the Azure c300 Gel Imaging System (Azure Biosystems Inc., Dublin, CA, USA). The Image-Pro Plus software (www.mediacy.com › 78-products › image-pro-plus) was used to analyze the gray value of the protein bands. GAPDH was selected as the internal reference to calculate the relative expression level of the samples.
2.9 Cell counting kit‑8 (CCK‑8) assay
CCK-8 (Kangrun Biology, China) was used to assess the proliferation ability of the 16HBE cells. Five thousand 16HBE cells were seeded into 96-well plates. A 100 μl-DMEM medium was added to each well, and five duplicate wells were designed for each group. The 96-well plates were incubated for 24, 48, 72, and 96 h. Then, 10 μl of CCK-8 reagent was added to each well and incubated for 3 h at 37 °C. Finally, the optical density (OD) value at 450 nm was measured using a microtiter plate reader, and the absorbance was calculated.
2.10 Transwell assay
The transwell chamber (Corning Inc., Corning, NY., USA) was used to evaluate the migration (or cell invasive) ability of the 16HBE cells. The cells were resuspended in serum-free media and seeded into the upper chamber at a density of 4×10 3 cells /well, using a volume of 200 μl. An 800 μl culture solution with 30% FBS was subsequently added to the lower chamber. After incubation for 24 h, cells were fixed, stained, and visualized under an inverted microscope. Cells that migrated to the sublayer of the microporous membrane were counted. Five fields were selected for each sample to count the number of cells, and the mean number was obtained.
2.11 Double luciferase reporter gene assay
We predicted the binding sequences between lncRNA PTTG3P and miR-192-3p, miR-192-3p, and CCNB1 from the Starbase (http://starbase.sysu.edu.cn/). We designed and synthesized the pSI-Check2 vector (Hanbio Biotechnology Co., Ltd Shanghai, China) including the wild-type (WT) and mutant (MT) binding sites of miR-192-3p within the PTTG3P sequence or CCNB1 3′UTR; we then named the PTTG3P-WT/MT and CCNB1-WT/MT vectors, respectively. The synthesized vectors with miR-192-3p mimic or mimic-NC were transfected into the 16HBE cells for 48 h, and the dual-luciferase reporter system (Promega, USA) was used to determine the luciferase activity.
2.12 Statistical analysis
Data are expressed as mean ± standard deviation (X‾±SD) One-way analysis of variance and t-test were used to compare the differences between groups. A p<0.05 indicated statistical significance. GraphPad Prism 8.0 and R 4.0.3 were used for statistical analysis.