biomedicines Article Systemic Profiles of microRNAs, Redox Balance, and Inflammation in Lung Cancer Patients: Influence of COPD Liyun Qin 1 , Maria Guitart 1,2 , Víctor Curull 1,2 , Albert Sánchez-Font 1,2 , Xavier Duran 3 , Jun Tang 1,2 , Mireia Admetlló 1,2 and Esther Barreiro 1,2, * 1 Pulmonology Department-Muscle Wasting and Cachexia in Chronic Respiratory Diseases and Lung Cancer Research Group, IMIM-Hospital del Mar, Parc de Salut Mar, Health and Experimental Sciences Department (CEXS), Universitat Pompeu Fabra (UPF), Universitat Autònoma de Barcelona, Parc de Recerca Biomèdica de Barcelona (PRBB), 08003 Barcelona, Spain; liyun.qin@e-campus.uab.cat (L.Q.); mguitart@imim.es (M.G.); VCURULL@parcdesalutmar.cat (V.C.); ASanchezF@parcdesalutmar.cat (A.S.-F.); jun.tang2@e-campus.uab.cat (J.T.); madmetllo@parcdesalutmar.cat (M.A.) 2 Centro de Investigación en Red de Enfermedades Respiratorias (CIBERES), Instituto de Salud Carlos III (ISCIII), 08003 Barcelona, Spain 3 Scientific and Technical Department, Hospital del Mar-IMIM, 08003 Barcelona, Spain; xduran@imim.es * Correspondence: ebarreiro@imim.es; Tel.: +34-93-316-0385; Fax: +34-93-316-0410 Abstract: Lung cancer (LC) risk increases in patients with chronic respiratory diseases (COPD). MicroRNAs and redox imbalance are involved in lung tumorigenesis in COPD patients. Whether systemic alterations of those events may also take place in LC patients remains unknown. Our objectives were to assess the plasma levels of microRNAs, redox balance, and cytokines in LC  patients with/without COPD. MicroRNAs (RT-PCR) involved in LC, oxidized DNA, MDA-protein  adducts, GSH, TEAC, VEGF, and TGF-beta (ELISA) were quantified in plasma samples from non- Citation: Qin, L.; Guitart, M.; Curull, LC controls (n = 45), LC-only patients (n = 32), and LC-COPD patients (n = 91). In LC-COPD V.; Sánchez-Font, A.; Duran, X.; Tang, patients compared to controls and LC-only, MDA-protein adduct levels increased, while those of J.; Admetlló, M.; Barreiro, E. Systemic GSH decreased, and two patterns of plasma microRNA were detected. In both LC patient groups, Profiles of microRNAs, Redox miR-451 expression was downregulated, while those of microRNA-let7c were upregulated, and levels Balance, and Inflammation in Lung of TEAC and TGF-beta increased compared to the controls. Correlations were found between clinical Cancer Patients: Influence of COPD. and biological variables. A differential expression profile of microRNAs was detected in patients with Biomedicines 2021, 9, 1347. https:// LC. Moreover, in LC patients with COPD, plasma oxidative stress levels increased, whereas those of doi.org/10.3390/biomedicines9101347 GSH declined. Systemic oxidative and antioxidant markers are differentially expressed in LC patients Academic Editor: Alice M Turner with respiratory diseases, thus implying its contribution to the pathogenesis of tumorigenesis in these patients. Received: 7 September 2021 Accepted: 22 September 2021 Keywords: LC and COPD; microRNAs; prooxidants and antioxidants; GSH; inflammatory cytokines; Published: 29 September 2021 associations between clinical and biological variables Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- 1. Introduction iations. Lung cancer (LC) is still a leading cause of cancer-related mortality worldwide. Several etiologic factors contribute to LC, among which chronic obstructive pulmonary disease (COPD) is a major contributor [1–4]. In patients with COPD, particularly in emphysema phenotype, LC development was five times greater than in smokers with no COPD [4,5]. Copyright: © 2021 by the authors. Recently, our group and others have demonstrated the implications of relevant biological Licensee MDPI, Basel, Switzerland. mechanisms in the increased LC predisposition seen in patients with COPD [6–13]. This article is an open access article Adaptation to environmental factors and tumorigenesis are mediated through many distributed under the terms and different biological events including epigenetics. Epigenetic control of cellular processes conditions of the Creative Commons includes DNA methylation, histone acetylation and methylation, and chromatin remodel- Attribution (CC BY) license (https:// ing in tumor development and progression [14]. Furthermore, non-coding single-stranded creativecommons.org/licenses/by/ RNA molecules (microRNAs) have also been shown to regulate the cellular processes 4.0/). Biomedicines 2021, 9, 1347. https://doi.org/10.3390/biomedicines9101347 https://www.mdpi.com/journal/biomedicines Biomedicines 2021, 9, 1347 2 of 16 involved in lung tumorigenesis, such as cell proliferation and invasion, apoptosis, angio- genesis, and adaptation to hypoxia [15–17]. Moreover, patterns of microRNA expression may also be used in clinics, given their prognosis value [16,18]. In a previous study from our group [13], expression levels of the microRNAs miR-21, miR-200b, miR-210, and miR- let7c were increased in lung tumor samples from non-small cell LC (NSCLC) patients with COPD compared to those with no COPD. Whether levels of the same microRNAs may be differentially expressed in the plasma of LC patients with and without COPD remains to be seen. Increased oxidative stress and inflammatory events have also been shown to contribute to the greater predisposition of patients with COPD to develop lung tumors [19]. In this study [19], a differential expression profile of oxidative stress and inflammatory markers was found in the blood and tumors of patients with LC-COPD compared to patients with no COPD. Importantly, increased oxidative stress may also trigger the expression of several microRNAs under pathologic conditions [20]. Several cytokines and growth factors may also mediate lung tumorigenesis in patients with COPD [21,22]. Cellular processes such as apoptosis, repair, and angiogenesis can be mediated through the action of inflammatory molecules [21,22]. Furthermore, vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-beta have also been shown to promote lung tumorigenesis through increased tumor growth and metastasis in patients with underlying respiratory diseases [23–25]. Hence, we hypothesized that a differential expression profile of microRNAs known to be involved in lung tumorigenesis may be identified in the blood compartment of patients with LC and underlying COPD compared to those without COPD. Elucidation of those biological events may add insight into the mechanisms that render COPD patients more prone to develop LC. Moreover, associations with oxidative stress and inflammatory mark- ers were also assessed. Thus, our objectives were that, in plasma samples from LC patients with and without COPD, the following mechanisms were explored: (1) expression levels of microRNAs known to be involved in lung carcinogenesis; (2) redox balance, prooxidant and antioxidant markers; (3) VEGF and TGF-beta 1 protein levels; (4) relationships between clinical and biological variables; and (5) potential associations between expression levels of microRNAs and those of oxidative stress and inflammatory cytokines. A group of non-LC control subjects was also included in the current investigation. 2. Methods 2.1. Study Subjects This was a hospital-based study in which patients and control subjects were recruited consecutively for 10 years (2009–2019). For the investigation, 168 Caucasian patients were recruited in total. Specifically, 123 patients with NSCLC were recruited from the Lung Cancer Clinic of the Respiratory Medicine Department at Hospital del Mar (Barcelona, Spain). Ninety-one out of the 123 patients had underlying COPD and 32 patients had NSCLC with no COPD. A group of non-tumor control subjects (n = 45) were also recruited for the purpose of the investigation from the COPD Clinics at Hospital del Mar. Therefore, for the purpose of comparisons, the following study groups were established: (1) 45 subjects without LC (32 males, non-LC control group), (2) 32 patients with only LC (15 males, LC- only group), (3) 91 patients with LC and COPD (82 males, LC-COPD group). COPD was defined following existing guidelines [26,27]. Exclusion criteria for all the patients and control subjects included other chronic cardiovascular or respiratory disorders, chronic metabolic diseases, signs of severe bronchial inflammation and/or infection, current or recent invasive mechanical ventilation, chronic oxygen therapy, and poor tolerance and/or collaboration. Approval was obtained from the institutional Ethics Committee on Human Investigation (Hospital del Mar-IMIM, Barcelona, protocol # 2008/3390/I, 4 February 2008, following World Medical Association guidelines (Helsinki Declaration of 2008) for research on human beings. Informed written consent was obtained from all participants. Biomedicines 2021, 9, 1347 3 of 16 2.2. Clinical Assessment Nutritional evaluation included the assessment of body mass index (BMI) and blood analytical parameters in all participants. Lung function parameters were determined in all study subjects following standard procedures [11,13,19]. TNM staging [28,29] was determined only in all patients with LC. 2.3. Blood Samples In all the study patients and non-LC control subjects, blood samples were obtained from the arm vein after an overnight fasting period. Blood specimens were centrifuged at 1500× g for 15 min to collect the plasma samples, which were immediately frozen at −80 ◦ C until further analyses. 2.4. Quantification of microRNAs RNA isolation. Previously described methodologies were used for this set of experi- ments [13]. RNA was isolated from 500 microL plasma samples using 500 microL TRIzol reagent (Cat. 15596026, Thermo Fisher Scientific, Waltham, MA, USA). After incubation of the samples at room temperature for 10 min to achieve complete dissociation of nucleopro- tein complexes, 200 microL chloroform were added, and samples were then centrifuged at 13,500 rpm at 4 ◦ C for 15 min. The aqueous phase was recovered, and the RNA was precipitated with 600 microL isopropanol. Subsequently, samples were incubated at 4 ◦ C for 30 min and were then cooled down to −20 ◦ C overnight. After thawing the samples at room temperature, they were centrifuged at 13,500 rpm at 4 ◦ C for 10 min, and the supernatant was removed. The remaining pellet was then washed using one mL solution of 75% ethanol to be subsequently centrifuged at 9000 rpm at 4 ◦ C for five minutes. The RNA containing pellet was air-dried for 30 min and was then dissolved in 20 microL RNase-free water. To assess the quality and purity of the isolated RNA, concentrations of total RNA were determined using NanoDrop 1000 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. MicroRNA reverse transcription (RT). TaqMan® Advanced miRNA cDNA Synthesis Kit (Cat. A28007, Thermo Fisher Scientific, Waltham, MA, USA) was used to prepare cDNA templates following the manufacturer’s instructions. Total RNA isolated samples were manipulated to add a poly (A) tailing on the 30 position and an adaptor on the 50 position of the mature microRNAs. Initially, 3 microL Poly (A) reaction mix (0.5 microL Poly (A) buffer, 0.5 microL ATP, 0.3 microL Poly (A) enzyme, and 1.7 microL RNase-free water) was mixed with 2 microL of each sample. The mixture was then incubated in a thermal cycler (Geneamp PCR System 2400, Perkin Elmer, Waltham, MA, USA) to perform the polyadenylation reaction at 37 ◦ C for 45 min. This step was followed by incubation at 65 ◦ C for 10 min to stop the reaction. Immediately, the samples were supplemented with 10 microL ligation reaction mix (3 microL ligase buffer, 4.5 microL PEG 8000, 0.6 microL ligation adaptor, 1.5 microL RNA ligase, and 0.4 microL of RNase-free water) to incorporate the adaptor at the 50 position. Samples underwent standard cycling at 16 ◦ C for 60 min. MicroRNAs with both poly (A) tail and the adaptor were reverse transcribed (RT) to achieve cDNA. First, modified microRNA samples were mixed with 15 microL RT reaction mix (6 microL RT buffer, 1.2 microL dNTP mix, 1.5 microL universal RT primer, 3 microL RT enzyme mix, and 3.3 microL RNase-free water). Samples were subsequently incubated in the thermal cycler at 42 ◦ C for 15 min to perform the reverse transcription, and finally, they were incubated at 85 ◦ C for 5 min to stop the reaction. A cDNA amplification step was performed to increase the number of cDNA molecules. Forty-five microL of miR-Amp reaction mix (25 microL miR-Amp master mix, 2.5 microL miR-Amp primer mix, and 17.5 microL RNase-free water) from the synthesis kit were mixed with 5 microL RT reaction product. The amplification reaction consisted of different cycles: enzyme activation at 95 ◦ C for 5 min, denaturation at 95 ◦ C for three seconds, and finally, the extension of the cDNA at 60 ◦ C for 30 s. Denaturation and extension cycles were repeated 14 more times to ensure a sufficient quantity of cDNA. Subsequently, samples Biomedicines 2021, 9, 1347 4 of 16 were incubated at 99 ◦ C for 10 min to stop the reaction, and they were finally kept at −80 ◦ C up until the performance of the real-time polymerase chain reaction (PCR) procedures. Quantitative real time-PCR amplification (qRT-PCR). Real-time PCR was performed using specific primers for the target microRNAs in the study: miR-451, miR-210, miR- 126, miR-21, miR-let7c, miR-145, miR-200b, and miR-223 (Table 1). Taqman advanced microRNA 159a assay from Arabidopsis thaliana was used as an exogenous control in order to normalize the miRNA amplification. Briefly, five microL of the resulting cDNA samples were mixed with one microL of each specific primer, 10 microL TaqMan fast-advanced master mix (Cat. 4444964, Thermo Fisher Scientific), and four microL RNase-free water. The samples were run in a thermal cycler (QuantStudio system, Thermo Fisher Scientific). The first step was the enzyme activation, achieved at 95 ◦ C for 20 s, which was followed by 40 combined cycles of denaturation (95 ◦ C for one second) and final annealing (60 ◦ C for 20 s). Duplicates from all samples were run, and the average value was calculated for all the study samples. The results obtained from the experiments were collected and analyzed using the ExpressionSuite Software version 1.1 from Applied Biosystems (ThermoFisher Scientific), in which the comparative CT method (2−∆∆CT ) for relative quantification was used [30]. Table 1. MicroRNA assays used for the quantitative analyses of the target genes using real-time PCR. Assay Name Assay ID miRbase Accession Number hsa-miR-451a 478107_mir MIMAT0001631 hsa-miR-210-3p 477970_mir MIMAT0000267 hsa-miR-126-3p 477887_mir MIMAT0000445 hsa-miR-21-5p 477975_mir MIMAT0000076 hsa-let7c-5p 478577_mir MIMAT0000064 hsa-miR-145-5p 477916_mir MIMAT0000437 hsa-miR-200b-3p 477963_mir MIMAT0000318 hsa-miR-223-3p 477983_mir MIMAT0000280 ath-miR159a 478411_mir MIMAT0000177 Abbreviations: ID, identification; hsa, homo sapiens; miR, microRNA; MIMAT, mature microRNA; ath, arabidop- sis thaliana. 2.5. Quantification of Oxidative Stress Markers and Cytokines In a subset of representative individuals: 40 non-LC control subjects, 19 LC-only patients, and 20 LC-COPD patients, markers of oxidative stress and cytokines were also analyzed in the blood samples. Oxidatively damaged DNA. Levels of oxidative DNA adduct 8-hydroxy-2-deoxy guano- sine (8-OHdG) were measured in plasma using the DNA Damage (8-OHdG) ELISA kit (StressMarq Biosciences INC ., Victoria, BC, Canada) following the specific manufacturer’s in- structions and previously described methodologies [13,19]. Briefly, 50 microL of plasma was incubated with 50 microL of antibody per well at room temperature for one hour in a plate cover. After several washes, samples were incubated with 3,30 ,5,50 -tetramethylbenzidine (TMB) substrate in the dark at room temperature for 30 min. Immediately afterwards, 100 microL of stop solution was poured into each well. Samples were then shaken from side to side and thoroughly mixed with the solution. After terminating this reaction, the absorbance was read at 450 nm in all the sample wells. A standard curve was always generated with each assay run. Intra-assay coefficients for all the samples ranged from 0.17% to 9.80%. The minimum detectable concentration of DNA in plasma was set to be 0.94 ng/mL (StressMarq Biosciences INC , Victoria, BC, Canada). Malondialdehyde (MDA)-protein adducts. Levels of MDA-protein adducts were mea- sured in plasma using the OxiSelectTM MDA Adduct Competitive ELISA Kit (Cell Biolabs, Inc., San Diego, CA, USA) following the specific manufacturer’s instructions and previ- ously described methodologies [13,19]. First, an MDA conjugate was coated on an ELISA plate, then 50 microL of plasma specimens were added to the MDA conjugate preabsorbed ELISA plate and incubated at room temperature for 10 min on an orbital shaker. After a Biomedicines 2021, 9, 1347 5 of 16 brief incubation, the primary antibody was added and incubated at room temperature for one hour on an orbital shaker. After three washes, samples were incubated with secondary antibody at room temperature for another hour on an orbital shaker. After three washes, the substrate solution was added at room temperature for 20 min, and samples were again shaken on an orbital shaker. Immediately afterwards, 100 microL of the stop solution was poured into each well. Samples were then thoroughly mixed with the solution. After completing this reaction, the absorbances were read at 450 nm. A standard curve was always generated with each assay run. Intra-assay coefficients of variation for all the sam- ples ranged from 0.11% to 9.73%. The minimum detectable concentration of MDA-protein adducts in plasma was set to be 6 pmol/mL (Cell Biolabs, Inc., San Diego, CA, USA). Reduced glutathione (GSH). GSH was measured in the blood using the Human reduced glutathione (GSH) ELISA Kit (MyBioSource, San Diego, CA, USA) following the specific manufacturer’s instructions and previously described methodologies [11,19]. Fifty microL samples were added to every sample well and incubated with horseradish (HRP)-conjugate reagent at 37 ◦ C for 60 min. The plate was covered with a closure plate membrane during the experiment. After four washes, 50 microL chromogen solution A and 50 microL chromogen solution B were added to each well, and samples were then incubated at 37 ◦ C in the dark for 15 min. Finally, 50 microL of the stop solution were poured into each well. The absorbance in each sample was read at 450 nm. Intra-assay coefficients for all the samples ranged from 0.15% to 9.93%. The minimum detectable concentration of GSH in plasma was set to be 1.56 µmol/L (MyBioSource, San Diego, CA, USA). Plasma levels of Trolox Equivalent Antioxidant Capacity (TEAC). TEAC levels were deter- mined using the OxiSelectTM Trolox Equivalent Antioxidant Capacity (TEAC Assay Kit (ABTS, Cell Biolabs, Inc., San Diego, CA, USA) following the manufacturer’s instructions. Twenty-five microL samples were added to the microplate well, and upon addition of 150 microL of the diluted 2,20 -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) reagent, samples were mixed thoroughly. Samples were then incubated on an orbital shaker for five minutes. Finally, the absorbance was read at 405 nm in all the sample wells. An- tioxidant activity was determined by comparison with the Trolox standards. Intra-assay coefficients of variation for all the samples ranged from 0.03% to 9.75%. (Cell Biolabs, Inc., San Diego, CA, USA). The minimum detectable concentration of TEAC in plasma was set to be 250.29 g/mol (Cell Biolabs, Inc., San Diego, CA, USA). Cytokines. Protein levels of the cytokines TGF-beta 1 and VEGF-A were quantified using specific ELISA kits (RayBiotech, Norcross, GA, USA) for each cytokine following the manufacturer’s instructions and previously described methodologies [13,31,32]. All samples were incubated with the specific primary antibodies and were always run together in each assay. Before commencing the assay, samples and reagents were equilibrated to room temperature. Standards (100 microL) were performed as per the manufacturer’s instructions. The protocol was followed according to the corresponding manufacturer’s instructions for each cytokine. Intra-assay coefficients of variation for all the samples ranged from 0.13% to 9.66% (TFG-beta1) and from 0.13% to 9.93% (VEGF). Absorbances were read at 450 nm in all sample wells. A standard curve was always generated with each assay run. The minimum detectable concentration of TGF-beta 1 was 18 pg/mL (RayBiotech, Norcross, GA, USA). The minimum detectable concentration of VEGF-A was 3.59 pg/mL (RayBiotech, Norcross, GA, USA). 2.6. Statistical Analysis Normality of the study variables was tested using the Shapiro–Wilk test. Data are expressed as mean and standard deviation (SD) in tables and figures. MicroRNA-451 was selected as the target variable to calculate the sample size. Having a priori unbalanced design, where the LC-COPD group had twice as many patients as the LC group and the control group, a minimum sample of 27 non-LC controls, 27 LC, and 54 LC-COPD patients was required to achieve an 80% statistical power, taking a within groups mean square error equals to 4393. Statistical significance was established at p < 0.05. Potential differences of Biomedicines 2021, 9, 1347 6 of 16 quantitative variables among the study groups were assessed using the one-way analysis of variance (ANOVA) and Tukey’s post hoc analysis to adjust for multiple comparisons. Chi- square test was employed to assess potential differences in categorical variables (smoking history) among the three study groups. Comparisons of the results obtained from the micro- RNA expression analyses within each study group were determined using the Duncan multiple comparison test. Correlations between clinical and biological variables were explored using the Pearson’s correlation coefficient. Bivariate analysis was performed to test associations between two variables for the study population. All statistical analyses were performed using the software SPSS 23.0 (SPSS Inc, Chicago, IL, USA). 3. Results 3.1. Clinical Characteristics Anthropometric variables such as age, body weight, and BMI did not significantly differ among the study subjects (Table 2). The proportions of active smokers were similar in the three study groups (Table 2). However, the proportions of ex-smokers were significantly greater in the LC-COPD group than in the LC-only group and showed a tendency to be higher than in the control subjects (p = 0.1, Table 2). The proportions of never smokers were significantly lower in the LC-COPD patients than in the LC-only and the control subjects (Table 2). No significant differences were seen in the number of packs-year among the study groups (Table 2). LC-COPD patients had moderate airway obstruction compared to LC-only and the controls, who had normal lung function parameters (Table 2). LC staging was similar between the two study groups (Table 2). In both groups of patients, nutritional status was preserved, and globular sedimentation velocity (GSV) was increased compared to the controls (Table 2). For both groups of patients, the comorbidities and different treatments are illustrated in Table 2. No significant correlations were found between any of the comorbidities and treatments with the study biological markers. Table 2. Clinical characteristics of the study patients. Non-LC Controls LC-Only LC-COPD N = 45 N = 32 N = 91 Anthropometry Age (years) 63 (10) 64 (11) 67 (9) Male, N/female, N 32/13 15/17 82/9 Body weight (kg) 75 (16) 70 (14) 72 (12) BMI (kg/m2 ) 27 (5) 26 (4) 25 (4) Smoking history Active, N (%) 15 (33) 11 (34) 39 (43) Ex-smoker, N (%) 17 (38) 9 (28) 48 (53) # Never smoker, N (%) 13 (29) 12 (38) 4 (4) ***,### Packs-year 47 (26) 46 (15) 58 (21) Lung function testing FEV1 , % predicted 80 (24) 94 (15) *** 62 (14) ***,### FEV1 /FVC, % predicted 70 (12) 78 (6) *** 61 (9) ***,### DLCO , % predicted 81 (22) 82 (19) 68 (19) **,## KCO , % predicted 82 (22) 82 (15) 75 (21) TNM staging Stage 0-II: N (%) NA 20 (62) 53 (58) Stage III: N (%) NA 7 (22) 19 (21) Stage IV: N (%) NA 5 (16) 19 (21) Biomedicines 2021, 9, 1347 7 of 16 Table 2. Cont. Non-LC Controls LC-Only LC-COPD N = 45 N = 32 N = 91 Blood parameters Total leukocytes/µL 8.2 (4.1) × 103 10.5 (4.2) × 103 * 9.7 (3.6) × 103 Total neutrophils/µL 5.3 (2.8) × 103 8.3 (4.4) × 103 *** 7.2 (3.5) × 103 * Total lymphocytes/µL 2.1 (1.1) × 103 1.4 (0.6) × 103 1.9 (3.0) × 103 Albumin (g/dL) 4.4 (0.6) 4.4 (0.9) 4.0 (0.6) **,# Total proteins (g/dL) 7.3 (0.8) 7.0 (1.0) 7.1 (0.8) CRP (mg/dL) 4.1 (8.2) 5.3 (9.4) 5.7 (7.8) Fibrinogen (mg/dL) 451 (147) 463 (131) 481 (157) GSV (mm/h) 16.3 (13.9) 27.6 (15.3) * 33.4 (22.0) ** Comorbidities Hypertension, N (%) NA 10 (31.3) 45 (49.5) Type 2 Diabetes mellitus, N (%) NA 2 (6.3) 17 (18.7) Dyslipidemia, N (%) NA 2 (6.3) 26 (28.6) Treatments Diuretic, N (%) NA 5 (15.6) 20 (22.2) Angiotensin converting enzyme NA 2 (6.3) 6 (28.6) inhibitors, N (%) Angiotensin-2 receptor blockers, NA 2 (6.3) 14 (15.4) N (%) Beta blockers, N (%) NA 3 (9.4) 5 (5.5) Calcium channel blockers, N (%) NA 2 (6.3) 7 (7.7) HMG-CoA-reductase, N (%) NA 2 (6.3) 17 (18.7) Biguanides, N (%) NA 2 (6.3) 26 (28.6) LAMA, N (%) NA NA 60 (65.9) LABA, N (%) NA NA 30 (33) Inhaled corticosteroids, N (%) NA NA 17 (18.7) Values are expressed as mean (standard deviation). Abbreviations: COPD, chronic obstructive pulmonary disease; N, number of patients; m, meters; BMI, body mass index; FEV1 , forced expiratory volume in one second; DLco, carbon monoxide transfer; KCO , Krough transfer factor; g, grams; TNM, tumor, nodes, metastasis; NA, not applicable; dL, deciliter; mg, milligrams; CRP, C-reactive protein; GSV, globular sedimentation velocity; LAMA, long-acting muscarinic antagonists; LABA, long-acting beta-agonists; mm, millimeters; h, hour. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001 between any study group compared to non-LC controls; # p < 0.05; ## p < 0.01; ### p < 0.001 between LC-COPD group compared to LC-only group. 3.2. Differential Pattern of Systemic microRNA Expression in LC Patients Compared to the non-LC controls, the plasma levels of miR-451 significantly decreased in both groups of patients (Figure 1). Systemic levels of miR-210 significantly declined in LC-COPD compared to LC-only patients (Figure 1). The plasma levels of miR-let7c were significantly greater in LC-only and LC-COPD patients than in the control subjects (Figure 1). Significant associations were detected between plasma microRNA-let7c expres- sion levels and total leukocytes and neutrophil counts (r = 0.467, p = 0.011, r = 0.498, and p = 0.006, respectively). No significant differences were seen in the plasma expression levels of miR-126, miR-21, miR-145, miR-200b, or miR-223 among the study groups (Figure 1). Biomedicines 2021, 9, x FOR PEER REVIEW 8 of 16 Biomedicines 2021, 9, x FOR PEER REVIEW 8 of 16 expression levels expression levels and and total total leukocytes leukocytes and and neutrophil neutrophil counts counts (r (r == 0.467, 0.467, pp == 0.011, 0.011, rr == 0.498, 0.498, Biomedicines 2021, 9, 1347 and p = 0.006, respectively). No significant differences were seen in the plasma expression and p = 0.006, respectively). No significant differences were seen in the plasma expression8 of 16 levels of levels of miR-126, miR-126, miR-21, miR-21, miR-145, miR-145, miR-200b, miR-200b, oror miR-223 miR-223 among among the the study study groups groups (Fig- (Fig- ure 1). ure 1). Meanvalues Figure 1.1.Mean Figure valuesandandstandard standard deviation deviation (relative (relative expression) expression) of microRNA of microRNA microRNA (miR)(miR) expression expression Figure 1. Mean values and standard deviation (relative expression) of (miR) expression in in plasma plasma samples samples ofof non-LC non-LC controls controls (white (white bars), bars), LC-only LC-only patients patients in plasma samples of non-LC controls (white bars), LC-only patients (grey bars), and LC-COPD (grey (grey bars), bars), and and LC-COPD LC-COPD patients (black patients(black patients bars). (blackbars). Statistical bars).Statistical Statisticalsignificance: significance: significance: * p < 0.05 * p*