Lung tissue Acquirement
This study was approved by the Institutional Ethics Committee of the Second Xiangya Hospital of Central South University. We recruited consecutive patients from April 2018 to September 2019. Human lung tissue from patients with COPD (n = 10) and controls (n = 10) was obtained from patients undergoing surgery and who gave informed consent. All the 20 patients were enrolled with primary lung cancer of stage 1 and had the surgery of pulmonary segmentectomy or lobectomy. All the patients were clinically stable for 4 weeks without acute pulmonary infection, did not receive chemotherapy half a year before the study, and did not have obstructive atelectasis, metastasis, other pulmonary diseases, and severe diseases in other systems. The lung tissue 5 cm away from tumor margin was used in our studies. The pathological examination confirmed that these samples presented lung structure without metastasis or inflammation.
Cell lines and cell culture
HBE cells were cultured in RPMI 1640 (Gibco, C11875500BT) supplemented with 10% fetal bovine serum (Gibco, 10082147), 100U/ml penicillin and 100 µg/ml streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) in 5% CO2 atmosphere at 37°C. Cells were passaged at 80% confluence and grown to full confluence for the experiments. All experiments were performed in triplicate and repeated independently at least three times. After serum starvation for 24 h, the HBE cells were treated with CSE at the indicated concentrations.
Preparation of CSE
CSE was prepared as previously described [10]. Briefy, one nonfiltered Fu-Rong cigarette (Furong, Changde Cigarette Company, Hunan, China) was burned, and the smoke was passed through 20 ml of phosphate-bufered saline via a vacuum pump. This 100% CSE solution was adjusted to 7.2–7.4 and filtered through a 0.22 m membrane filter to remove large particles and bacteria before use. Then, CSE was diluted with PBS to obtain concentrations of 1%, 2.5%, and 5%. CSE was freshly prepared within the 30 min preceding each experiment.
Quantitative Real-Time polymerase chain reaction (qRT-PCR)
Total RNA was isolated and extracted from different groups of lung tissues and cells by adding TRIzol (Life Technologies, Carlsbad, CA), chloroform, isopropanol, and 75% ethanol. Single-stranded cDNA was synthesized from 1µg RNA using a reverse reaction kit (Thermo Fisher Scientific. Waltham, MA, USA). Then qRT-PCR was performed on an ABI 7500 instrument (Applied Biosystems, Foster City, CA) using iTapTM SYBR Green Supermix with ROX dye (Bio-Rad Laboratories, Hercules, CA). The primers for miR-152-3p and U6 were obtained from RiboBio Co. Ltd. (Guangdong, China). The forward and reverse primers for other genes (RiboBio, Guangzhou, China) are listed as follows:
CCAT1, forward 5’-CACCTACGCATACCTCTGCTTC-3’, reverse 5’-TGATTGCTCCTGTTTCCCTTTG-3’; IL-1β, forward 5’-GAAACCCTCTGTCATTCGCTC-3’, reverse 5’-CAGACACTGCTACTTCTTGCCC-3’; IL-6, forward 5’-TGCCAGCCTGCTGACGAA-3’, reverse 5’-AGCTGCGCAGAATGAGATGA-3’; GAPDH, forward 5’-GAACGGGAAGCTCACTGG-3’, reverse 5’- GCCTGCTTCACCACCTTCT-3’.
Cell Transfection
miR-152-3p mimic, miR-152-3p inhibitor, mimic negative control (mimic-NC), inhibitor negative control (inhibitor-NC), small interfering RNAs (siRNAs) against CCAT1 (si-CCAT1) and negative control siRNA (si-NC) were purchased from RiboBio (RiboBio, Guangzhou, China). CCAT1-overexpression vector (CCAT1) and empty vector (CCAT1-NC) were available from GeneChem (Shanghai, China). They were transfected into HBE cells by Lipofectamine 3000 (Thermo Fisher Scientific. Waltham, MA, USA), according to the manufacturer’s protocols.
Western blot analysis
Total proteins from cells were extracted using RIPA lysis buffer (Beyotime, Shanghai, China). Protein concentrations of all groups were determined by a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Proteins were separated by 10% SDS-PAGE, then transferred to PVDF membranes and incubated with specific primary antibodies against t-ERK (ab184699, Abcam, Cambridge, UK), p-ERK (ab201015, Abcam, Cambridge, UK), and β-actin (10068-1-AP, Proteintech, Wuhan, China) overnight at 4°C. The membrane was incubated with HRP-labelled secondary antibody (Proteintech, Wuhan, China) for 1 h at room temperature. An enhanced chemiluminescence detection system (BIO-RAD, California, USA) was used to detect the protein bands.
Enzyme-Linked Immunosorbent Assay (ELISA)
Following appropriate CSE treatment and indicated transfection, the concentrations of interleukin-1β (IL-1β) and IL-6 from the culture supernatants of HBE cells were assayed using commercial the ELISA kits (Proteintech, Wuhan, China) referring to the manufacturer’s protocols. The results represent as the average of three independent replicates.
Dual-Luciferase Reporter Assay
The CCAT1 fragment with the predicted binding site of miR-152-3p was synthesized and cloned into the luciferase reporter gene to form the reporter vector CCAT1-wild-type (CCAT1-Wt). CCAT1-miR-152-3p binding site was mutated as instructed and named CCAT1 mutant (CCAT1-Mut). MiR-152-3p mimic and mimic-NC were co-transfected with CCAT1-Wt or CCAT1-Mut into HEK293T cells, respectively. After 48 h, the relative luciferase activity of each well was measured using the Dual Luciferase Reporter System (Pro-mega, Madison, WI, United States).
Statistical analysis
Quantitative data were presented as mean ± SD taken from at least three independent experiments. For relative gene expression, the mean value of the vehicle control group was defined as 1 or 100%. SPSS 21.0 (SPSS, Chicago, IL, USA) software was used for statistical analysis. Student’s t-test and one-way analysis of variance were used for the comparison of the statistical differences between groups. Tukey’s test or Dunnett’s T3 test was used for post hoc multiple comparisons according to the homogeneity test of variance. Significance was defined as p < 0.05.