All COPD subjects underwent clinical assessment including medical history, physical examination and symptoms evaluation using the modified Medical Research Council (mMRC) dyspnoea scale and COPD Assessment Test (CAT). Functional assessments included spirometry and six minute walk test (6MWT). On the basis of available data, body mass index, airway obstruction, dyspnoea, and exercise capacity (BODE) index for each of the COPD subjects was also calculated . To evaluate disease severity recruited COPD subjects were classified into four groups A-D according to the GOLD 2017 guidance . All study subjects underwent the sputum induction procedure and blood draw sampling for further serum and sputum miRNA profiling.
3.1 The mMRC dyspnea scale
mMRC is a five-level rating scale based on the patient’s perception of dyspnoea in daily activities. It consists of five statements that describe almost the entire range of dyspnoea from none (Grade 0) to almost complete incapacity (Grade 4) .
3.2 The CAT
CAT is a simple, health status instrument for patients with COPD . The self-administered questionnaire consists of eight items assessing various manifestations of COPD aiming to provide a simple quantified measure of health related quality of life. A decrease in CAT score represents an improvement in health status, whereas an increase in CAT score represents a worsening in health status. This questionnaire has been incorporated into the GOLD guidelines combined multidimensional assessment of specific patient attributes as a means of establishing a symptomatic threshold to guide stratification of the disease management .
Spirometry assessment was performed using a Lungtest 1000 spirometer (MES, Cracow, Poland) according to ATS/ERS guidelines . FEV1 (forced expiratory volume in 1 second), FVC (forced vital capacity) and FEV1/FVC% were evaluated. Parameters were presented as % of predicted value.
3.4 The 6 MWT
The 6MWT is used for evaluation of functional exercise capacity in patients with chronic respiratory diseases. In our study, 6 MWT was performed using the methodology specified by the Polish Respiratory Society guidelines. . Briefly, all COPD patients were instructed to walk as far as possible during 6 minutes. The 6MWT was performed in a flat, long, covered corridor which was 30 meters long, meter-by-meter marked. When the test was finished, the distance covered was calculated.
3.5 BODE index
This multidimensional scoring system for COPD patients evaluates the body mass index (BMI), measurement of airflow obstruction (FEV1% predicted), dyspnoea score (grade in mMRC scale), and exercise capacity (distance covered in 6MWT). This composite marker of disease takes into consideration the systemic nature of COPD and is used to predict long-term outcomes in this population .
3.6 Sputum induction
The sputum induction procedure was performed by a trained technician using the method described previously . Briefly, after salbutamol pretreatment (400 µg), aerosols of hypertonic saline (3%, 4% and 5%) were each inhaled for 7 min via ultrasonic nebulizer (DeVilbiss Ultraneb 3000, Sunrise Medical Ltd, USA) with an output of 1ml/min. Patients were asked to cough into a container after each cycle. The procedure was monitored by spirometry assessments at baseline and after each saline inhalation. If there was a fall in FEV1 of ≥ 20% versus baseline, the procedure was discontinued. Fall in FEV1 of 10–19% was an indication to continue the induction with the same concentration of saline.
3.7 Sputum analysis
The sputum was selected from the expectorate and processed within 2 hours as described previously [16–17]. Selected sputum plugs were dispersed using dithiothreitol (DTT), then the suspension was filtered and a total cell count of leukocytes and viability was assessed. Cytospins were stained with May-Grünwald-Giemsa. Differential cell counts were performed on 400 non-squamous cells. Sputum leukocytes were then used for further molecular evaluations.
3.8 RNA isolation
RNA isolation was performed using a mirVana™ miRNA Isolation Kit (Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s protocol. The quality and quantity of isolated RNA was assessed spectrophotometrically (Eppendorf BioPhotometrTM Plus, Eppendorf, Hamburg, Germany). The purity of total RNA was determined using RNA Nano Chips LabChipplates (ratio of 16S to 18S fraction) and RNA/miRNA was evaluated by automated electrophoresis using RNA Pico and RNA Nano Chips LabChipplates in an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
3.9 RNA expression analysis
cDNA was transcribed from 100 ng of total RNA, using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA) in a total volume of 20 µl. Reverse transcription (RT) master mix contained the following: 10 x RT buffer, 259 dNTP Mix (100 mM), 10 x RT Random Primers, MultiScribeTM Reverse Transcriptase, RNase Inhibitor, and nuclease-free water. The RT reaction was performed in a personal thermocycler (Eppendorf, Hamburg, Germany) in the following conditions: 10 min at 25°C, followed by 120 min at 37°C; then the samples were heated to 85°C for 5 s, and held at 4°C. The relative expression analysis was performed in 7900HT Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA) using TaqMan probes for the studied genes: IL-6 (Hs00174131_m1), IL6ST (Hs00174360_m1), STAT3 (Hs00374280_m1), and PIAS3 (Hs00966035_m1). The PCR mixture contained cDNA (1–100 ng), 20 x TaqManR Gene Expression Assay, 2 x KAPA PROBE Master Mix (2x) ABI Prism Kit (Kapa Biosystems, Wilmington, MA) and RNase-free water in a total volume of 20 µl. The expression levels (RQ values) of the studied genes were calculated using the delta-delta CT method, with the adjustment to the β-actin expression level and in relation to the expression level of calibrator (Human Lung Total RNA Ambion®), for which RQ value was equal to 1.
3.10 miRNA expression analysis
cDNA was transcribed from 10 ng of total RNA using a TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA) in a total volume of 15 µl according to the manufacturer’s protocol. The relative expression of miRNAs was assessed by qPCR reactions using TaqMan probes for the studied miRNA (Applied Biosystems, Carlsbad, CA) according to the manufacturer’s protocol. The following microRNA probes were used for the study: hsa-miR-1 (UGGA AUGUAAAGAAGUAUGUA), hsa-miR-106b (UAAAGUGCUGACAGUGCAGAU), hsa-miR-155 ( UUAAUGCUAAUCGUGAUAGGGGU). The PCR mixture contained 1.0 µl TaqMan MicroRNA Assay (20✕) 1.33 µl Product from RT reaction, 10.00 µl Ta q Man 2✕Universal PCR Master Mix, No AmpErase UNG, 7.76 µl Nuclease-free water (Applied Biosystems, Carlsbad, CA). The plates were placed in the Applied Biosystems 7900HT Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA) according to manufacturer’s protocol. The expression levels (RQ values) of the studied miRNAs were calculated using the delta-delta CT method, with the adjustment to the level of 001973 U6 snRNA expression (endogenous control) and to the expression of the calibrator (Human Lung Total RNA Ambion®), for which RQ value was equal to 1.
3.11 Statistical analysis
The Kruskal–Wallis test, Mann–Whitney U-test, Neuman–Keuls multiple comparison test, and Spearman’s rank correlation were used to assess the correlations between relative miRNA expression and spirometric parameters, GOLD classification, age and sex of patients. P = 0.05 was regarded as the level of statistical significance (StatSoft 12, Cracow, Poland).