A total of 34 patients who underwent lung surgery at Zhejiang Provincial People's Hospital (Hangzhou, China) from January 2014 to April 2015 were enrolled in the present study. These patients included 28 men and 6 women, ranging from 41–81 years old, with a median age of 66.5 years. None of the patients received preoperative chemotherapy, radiotherapy or intravenous corticosteroid therapy, and these patients did not exhibit failure of the heart, lung, brain or kidney, or any hematological diseases. Patients diagnosed with respiratory diseases other than COPD, including idiopathic pulmonary fibrosis and asthma, were excluded. According to the global strategy for the diagnosis of chronic obstructive pulmonary disease (1), these patients were sorted into 2 groups as follows: A control group (22 patients) and a COPD group (12 patients, exposed to cigarette smoke for >20 years). Lung function of each patient was examined by spirometry, which was performed by a trained technician according to the American Thoracic Society/European Respiratory Society guidelines (20). The lung tissues, taken from a distance of 5 cm from the negative margin of cancer tissues, were collected immediately following surgical resection and frozen instantly in liquid nitrogen or immersed in paraformaldehyde solution. The present study was approved by the Ethics Committee of Zhejiang Provincial People's Hospital (Hangzhou, China). Informed consent was obtained from all patients.

Lungs were immersed in paraformaldehyde for 24 h, then embedded in paraffin and cut into 4 µm thick sections. A total of 3 discontinuous paraffin-embedded sections from each lung tissue sample were stained with hematoxylin and eosin (H&E) in order to assess the morphological changes in the lungs. A total of 5 fields of view from each of the 3 sections from each lung sample were assessed using a light microscope (Olympus Corporation, Tokyo, Japan). The mean linear intercept (MLI), the mean alveolar airspace (MAA) and the number of alveolar counted per unit area (MAN) were obtained. MLI indicates the average distance between opposing walls of a single alveolus and is a measure of pulmonary airspace enlargement. MAA is also a measure of pulmonary airspace enlargement, while MAN is a measure of pulmonary airspace density.

SA-β-gal activity was assessed using an in situ β-galactosidase staining kit (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer's protocol. Lung tissues were fixed in β-galactosidase stationary solution for 15 min, then washed 3 times for 10 min each in PBS. Sections were then incubated with 1 ml staining solution mixture (10 µl staining solution A, 10 µl staining solution B, 930 µl staining solution C and 50 µl X-gal solution) for 2 h at 37°C. Following 3 washes with PBS, 5 fields of view from each of the 3 sections from each lung sample were examined using a light microscope (Olympus Corporation).

Total RNA was extracted from each lung sample using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. The final RNA purity and concentrations were determined using a spectrophotometer. cDNA was synthesized from the total RNA using the PrimeScript™ RT reagent kit with gDNA eraser (Takara Bio, Inc., Otsu, Japan) according to the manufacturer's protocol. qPCR analysis was performed using SYBR-Green qPCR kit (Takara Bio, Inc.) and the Agilent MX3000P qPCR system (Agilent Technologies, Inc., Santa Clara, CA, USA). The thermocycling conditions were as follows: Initial denaturation at 95°C for 15 min, followed by 45 cycles of denaturation at 95°C for 15 sec, annealing at 58°C for 15 sec, and elongation at 72°C for 1 min. The following primers were used (Invitrogen; Thermo Fisher Scientific, Inc.): SAL-RNA1, forward 5′-AGGCTGCCATCTCACCCTCATAC-3′ and reverse 5′-TCCTTACCTCTTCCCTTCCACCC-3′; SAL-RNA2, forward 5′-GGATGCTGTGAGCTTTGTGA-3′ and reverse 5′-GAAACCCCCAGAGCTGAGAC-3′; SAL-RNA3, forward 5′-ACTGCTGGGATAACGGTGAC-3′ and reverse 5′-TCTGTGCTCAGCTCTGCAAT-3′; SIRT1, forward 5′-GCAGATTAGTAGGCGGCTTG-3′ and reverse 5′-ACTTTCATCCTCCATGGGTTC-3′; p53, forward 5′-ACCACCATCCACTACAACTACAT-3′ and reverse 5′-CAGGACAGGCACAAACACG-3′; FOXO3a, forward 5′-TGGCAAGCACAGAGTTGGATGA-3′ and reverse 5′-TGGCGGGAGCGTGATGTTAT-3′; p21, forward 5′-ACTTTGATTAGCAGCGGAACA-3′ and reverse 5′-GAAAACAGTCCAGGCCAGTATG-3′; and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), forward 5′-TGAAGGTCGGAGTCAACGG-3′ and reverse 5′-CTGGAAGATGGTGATGGGATT-3′. All analyses were performed in triplicate. GADPH was used as a reference gene. The results were quantified using the 2^−ΔΔCq method (21).

Proteins were extracted from each lung sample by homogenizing the samples in ice-cold lysis buffer [50 mmol/l Tris (pH 7.4), 150 mmol/l NaCl, 0.1% sodium dodecyl sulfate, 1 mmol/l EDTA, 1% sodium deoxycholate, and 1% Triton X-100; Invitrogen; Thermo Fisher Scientific, Inc.] containing a protease inhibitor cocktail (1 mmol/l phenylmethylsulfonyl fluoride, 1 mg/l leupeptin and 1 mg/l aprotinin; Beyotime Institute of Biotechnology). The protein concentration in the samples was determined using a micro bicinchoninic acid protein assay kit (Pierce; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Next, equal amounts of protein (20 µg) from each sample were heated at 100°C for 5 min and then separated by electrophoresis on 8% sodium dodecyl sulfate-polyacrylamide gels. The separated proteins were electrotransferred to nitrocellulose membranes and blocked with Tris-buffered saline containing 0.1% Tween-20 (TBS-T) and 5% bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 2 h at room temperature. The membranes were then incubated with primary antibodies against p53 (#9282; 1:1,000 in TBS-T; Cell Signaling Technology, Inc., Danvers, MA, USA), p21 (sc-397; 1:500 in TBS-T; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), FoxO3a (#12829; 1:1,000 in TBS-T; Cell Signaling Technology, Inc.) SIRT1 (#2493; 1:1,000 in TBS-T; Cell Signaling Technology) and β-actin (ab8227; 1:1,000; Abcam, Cambridge, UK) overnight at 4°C on an orbital shaker. Following washing 3 times for 10 min each in TBS-T, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (#35552; 1:5,000 in TBS-T; Pierce; Thermo Fisher Scientific, Inc.) for 1 h at room temperature on an orbital shaker. Following 5 washes with TBS-T, immunoreactive bands on the membrane were detected using an enhanced chemiluminescence solution (GE Healthcare Life Sciences, Chalfont, UK), visualized by means of X-ray-film exposure, and analyzed using an UVP-GDS8000 gel-analysis system (Ultra-Violet Products, Ltd., Cambridge, UK). Protein expression levels were analyzed by densitometry and the values were normalized relative to those measured for β-actin, which was used as the loading control.

SIRT1 deacetylase activity was measured using the SIRT1 fluorimetric activity assay kit (Enzo Life Sciences, Inc., Farmingdale, NY, USA) according to the manufacturer's protocol. SIRT1 was immunoprecipitated as described previously (22). Following washing with PBS, Fluor de Lys substrate and NAD⁺ were added to the SIRT1-conjugated beads and incubated for 90 min at 37°C. Then, the substrate-SIRT1 mixture was placed on a white 96-well plate, and the Fluor de Lys developer II reagent was added to the 96 wells for 30 min at 37°C. Finally, the plate was analyzed by a microplate reading fluorimeter (M200; Tecan Group, Ltd., Männedorf, Switzerland) at 405 nm.

Statistical analyses were performed using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA). Data were expressed as the mean ± standard deviation. The significance of difference between groups was determined by means of independent-sample Student's t-tests. P<0.05 was considered to indicate a statistically significant difference.

The lung function of each patient was examined by spirometry. There were no significant differences between patients from the control group and the COPD group in forced vital capacity (FVC; P>0.05; Table I). However, the ratio of forced expiratory ventilation in 1 sec to FVC (FEV₁/FVC) and %FEV₁ in patients with COPD was significantly reduced compared with the control group (P<0.001; Table I).

The histological characteristics of lung tissues from the control group and the COPD group were analyzed by H&E staining. Compared with the control group (Fig. 1A), the airspace was visibly enlarged and the number of alveoli was decreased in lung tissues of the COPD group (Fig. 1B). The lung samples from the COPD group also exhibited merged alveoli and the formation of bullae, which was in accord with the pathological characteristics of COPD (Fig. 1B). Quantitative analyses of lung histomorphology revealed that the MLI and MAA of the COPD group was significantly higher compared with the control group (P<0.05; Table II) and the MAN was significantly decreased in the COPD group compared with the control group (P<0.05; Table II).

SA-β-gal activity was assayed in lung samples to analyze whether cellular senescence was exhibited in lung tissues of the patients with COPD. The cells in lung tissues with blue color were considered SA-β-gal positive, and so senescent. Compared with the control group (Fig. 2A), SA-β-gal activity was visibly increased in the COPD group (Fig. 2B) Most SA-β-gal-positive cells were located at the edges of alveoli (Fig. 2B).

The lncRNA expression of SAL-RNA1, SAL-RNA2 and SAL-RNA3 was measured by RT-qPCR. SAL-RNA2 and SAL-RNA3 mRNA expression levels were significantly increased in the lung tissues of the COPD group compared with the control group (P<0.05; Fig. 3). However, the SAL-RNA1 level in the COPD group was significantly decreased compared with the control group (P<0.05; Fig. 3).

p53 and p21 mRNA expression levels were significantly increased in lung tissues in the patients with COPD compared with the control group (P<0.05; Fig. 4). By contrast, SIRT1 and FoxO3a mRNA expression levels in the COPD group were significantly decreased compared with the control group (P<0.05; Fig. 4).

SIRT1, FoxO3a, p53 and p21 protein expression levels were visualized by western blotting. SIRT1 and FoxO3a protein levels were visibly reduced in the COPD group compared with the control group, while p53 and p21 protein levels were visibly upregulated in the COPD group compared with the control group (Fig. 5A). Accompanying the downregulation in SIRT1 protein levels, there was a significant reduction of SIRT1 activity in the COPD group compared with the control group (P<0.05; Fig. 5B).