In this study, a total of 258 patients with COPD and 311 matched non-COPD population controls were enrolled. The control subjects were healthy donors with Chinese Han descent. The subjects in both groups were self-reported Chinese Han individuals and were recruited from Wenzhou in the Zhejiang Province of China. Each subject was interviewed by interviewers who collected the patients’ demographic data and information related to risk factors, such as smoking cigarettes. The clinical analyses were performed at Wenzhou Medical University; diagnosis of patients was performed according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria. All patients were subjected for clinical testing and evaluation of genetic variations. Patients were excluded from this study if they had other respiratory diseases (bronchial asthma, bronchiectasis, cancer, or pulmonary tuberculosis). Control subjects were excluded if they had a history of lung disease, atopy, an acute pulmonary infection in the 4 weeks before the enrolment for this study. This study was approved by Institutional Review Board of Jilin Agricultural University and Ethics Committee of the Second Affiliated Hospital, Wenzhou Medical University. The investigator explained the purpose and risks of the study and provided the subject with a copy of the information sheet.
Based on the LD and haplotype block analysis using HaploReg 4, we included 68 SNPs in LD with the three tag SNPs with an r2 0.8 for an initial screening. Further bioinformatic analyses of the list of SNPs were performed to exclude predicted SNP motif changes less than 5. By applying the filter, 17 SNPs remained for further analysis. Because a SNP rs12905203 is expected to change 19 binding motifs for multiple transcription factors, we included this candidate functional variant along with the reported Asian COPD-associated variant for this genetic association study.
Isolation of genomic DNA from PBMCs was performed using a DNA extraction and purification kit (TAKARA, Dalian, China), according to the manufacturer’s instructions. Sequences of the PCR primers are listed in Additional file 3: Table S3. The genotypes at selected SNPs for each genomic DNA sample with PCR amplified target regions were determined using a TaqMan SNP genotyping assay kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). The assay followed the protocol in the manual, and PCR reactions were conducted on an OpenArray™ real-time PCR instrument (Applied Biosystems™, Foster City, CA, USA).
The demographic and clinical data between the COPD patients and the controls were analyzed using the chi-square test and Student’s t-test. Hardy-Weinberg equilibrium was calculated with a goodness of fit chi-squared test to assess the observed and expected genotype frequencies. The differences between the COPD patients and the control subjects in the context of the genotypes were analyzed using one-way analysis of variance and logistic regression for multivariate analysis. Age, sex, and smoking were used as covariates in the multivariate analyses. Statistical analyses were conducted using SPSS version 21.0 and Microsoft Excel.
Antibodies, plasmid DNAs, and cell lines
Expression constructs expressing human c-Fos (pLX304-FOS-V5) and c-Jun (pCLXSN-c-JUN) were purchased from Addgene (Addgene Headquarters, Cambridge, MA, USA). Luciferase activity assay backbone DNA pGLuc-mini-TK was purchased from New England Biolabs (Ipswich, MA, USA). Anti-KGF, anti-c-Fos, and anti-c-Jun antibodies were purchased from Abcam (Abcam, Inc. Shanghai, China). Sixty-nine skin fibroblast cell lines were established from healthy Chinese donors.
RNA isolation and quantitative RT-PCR
Total RNA from fibroblast cell lines was isolated using a Total RNA isolation and purification kit (Invitrogen Inc., Carlsbad, CA, USA) according to the manufacturer’s instructions. cDNA from each sample was synthesized using iScript cDNA Synthesis Kits (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Quantitative RT-PCR assays were performed using Power SYBR Green Master Mixture (Applied Biosystems™, Foster City, CA, USA) to determine the mRNA expression of the FGF7 gene in each sample, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal control. Fold changes were calculated according to the ΔΔCT method.
Electrophoretic mobility shift assays
Fifty base pair non-risk or risk DNA probes were synthesized and end-labeled with a Biotin-DNA labeling kit (Thermo-Fisher). Nuclear protein extracts were prepared from fibroblast cells transfected with plasmid DNA expressing c-Fos, c-Jun, or control vector. A portion of the cells was stimulated with PMA/Ionomycin (50 ng/ml, 500 ng/ml) for 2 h. Incubation of nuclear proteins with labeled probes was performed for 25 min at 37 °C in the binding buffer (1 μg poly dI-dC, 20 mM HEPES, 10% Glycerol, 100 mM KCl, and 0.2 mM EDTA, pH 7.9). DNA-protein complexes were resolved on non-denaturing acrylamide gels.
ChIP and qPCR
ChIP assays were performed using the Millipore Magna ChIP A kit (Millipore, Billerica, CA, USA) according to the manufacturer’s instructions. The chromatin-protein complexes were immune-precipitated by antibodies specific for c-Fos, c-Jun, or rabbit IgG (negative control) overnight at 4 °C. DNA was eluted from the immune-precipitated chromatin complexes, reverse-crosslinked, purified by a DNA purification kit (TAKARA, Dalian, China), and subjected to real-time PCR analysis. Sequences of primers are listed in the Additional file 4: Table S4
We cloned 200 bp of DNA sequence surrounding the variant rs12905203 into a minimal TK promoter luciferase plasmid. Each plasmid was transiently co-transfected with a pGL3-promoter transfection efficiency control plasmid for normalization. Luciferase assays were performed in fibroblast cells. Forty eight hours post-transfection, cells were treated with 50 ng/ml PMA/500 ng/ml Ionomycin for additional 48 h. The cells were lysed using a lysis butter provided by the Kit, and the luciferase activity was measured using the Luciferase Assay System (Promega, Madison, MA, USA).