International Journal of Molecular Sciences Article Analysis of Differentially Expressed MicroRNAs in Serum and Lung Tissues from Individuals with Severe Asthma Treated with Oral Glucocorticoids Marta Gil-Martínez 1,2,† , Clara Lorente-Sorolla 1,† , José M. Rodrigo-Muñoz 1,2 , Miguel Ángel Lendínez 1 , Gonzalo Núñez-Moreno 3,4,5 , Lorena de la Fuente 3 , Pablo Mínguez 3,4,5 , Ignacio Mahíllo-Fernández 6 , Joaquín Sastre 2,7 , Marcela Valverde-Monge 2,7 , Santiago Quirce 2,8 , María L. Caballero 2,8 , Francisco J. González-Barcala 2,9 , Ebymar Arismendi 2,10 , Irina Bobolea 2,10 , Antonio Valero 2,10 , Xavier Muñoz 2,11 , María Jesús Cruz 2,11 , Carlos Martínez-Rivera 2,12 , Vicente Plaza 2,13 , José M. Olaguibel 2,14 and Victoria del Pozo 1,2,15, * 1 Immunoallergy Laboratory, Immunology Department, Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Av. Reyes Católicos 2, 28040 Madrid, Spain 2 CIBER de Enfermedades Respiratorias (CIBERES), Instituto de Salud Carlos III (ISCIII), 28029 Madrid, Spain 3 Department of Genetics, Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), 28040 Madrid, Spain 4 Center for Biomedical Network Research on Rare Diseases (CIBERER), Instituto de Salud Carlos III (ISCIII), 28029 Madrid, Spain 5 Bioinformatics Unit, Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), 28040 Madrid, Spain 6 Biostatistics and Epidemiology Unit, Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD), 28040 Madrid, Spain 7 Allergy Department, Hospital Universitario Fundación Jiménez Díaz, 28040 Madrid, Spain 8 Department of Allergy, Hospital Universitario La Paz, IdiPAZ, 28046 Madrid, Spain Citation: Gil-Martínez, M.; 9 Pulmonology Department, Complejo Hospitalario Universitario de Santiago, Lorente-Sorolla, C.; Rodrigo-Muñoz, 15706 Santiago de Compostela, Spain J.M.; Lendínez, M.Á.; Núñez-Moreno, 10 Allergy Unit & Severe Asthma Unit, Pulmonology and Allergy Department, Hospital Clínic, G.; de la Fuente, L.; Mínguez, P.; 08036 Barcelona, Spain 11 Pulmonology Department, Hospital Vall d’Hebron, 08035 Barcelona, Spain Mahíllo-Fernández, I.; Sastre, J.; 12 Pulmonology Department, Hospital Germans Trias i Pujol, 08916 Badalona, Spain Valverde-Monge, M.; et al. Analysis 13 Respiratory Medicine Department, Hospital de la Santa Creu i Sant Pau, 08041 Barcelona, Spain of Differentially Expressed 14 Severe Asthma Unit, Department of Allergy, Complejo Hospitalario de Navarra, 31008 Pamplona, Spain MicroRNAs in Serum and Lung 15 Department of Medicine, Faculty of Medicine, Universidad Autónoma de Madrid, 28029 Madrid, Spain Tissues from Individuals with Severe * Correspondence: vpozo@fjd.es Asthma Treated with Oral † These authors have contributed equally to this work. Glucocorticoids. Int. J. Mol. Sci. 2023, 24, 1611. https://doi.org/10.3390/ Abstract: Nowadays, microRNAs (miRNAs) are increasingly used as biomarkers due to their po- ijms24021611 tential contribution to the diagnosis and targeted treatment of a range of diseases. The aim of Academic Editor: Anagha Malur the study was to analyze the miRNA expression profiles in serum and lung tissue from patients Received: 19 December 2022 with severe asthma treated with oral corticosteroids (OCS) and those without OCS treatment. For Revised: 4 January 2023 this purpose, serum and lung tissue miRNAs of OCS and non-OCS asthmatic individuals were Accepted: 11 January 2023 evaluated by miRNAs-Seq, and subsequently miRNA validation was performed using RT-qPCR. Published: 13 January 2023 Additionally, pathway enrichment analysis of deregulated miRNAs was conducted. We observed altered expression by the next-generation sequencing (NGS) of 11 miRNAs in serum, of which five (hsa-miR-148b-3p, hsa-miR-221-5p, hsa-miR-618, hsa-miR-941, and hsa-miR-769-5p) were validated by RT-qPCR, and three miRNAs in lung tissue (hsa-miR-144-3p, hsa-miR-144-5p, and hsa-miR-451a). Copyright: © 2023 by the authors. The best multivariate logistic regression model to differentiate individuals with severe asthma, treated Licensee MDPI, Basel, Switzerland. and untreated with OCS, was to combine the serum miRNAs hsa-miR-221-5p and hsa-miR-769-5p. This article is an open access article Expression of hsa-miR-148b-3p and hsa-miR-221-5p correlated with FEV1 /FVC (%) and these altered distributed under the terms and miRNAs act in key signaling pathways for asthma disease and the regulated expression of some genes conditions of the Creative Commons Attribution (CC BY) license (https:// (FOXO3, PTEN, and MAPK3) involved in these pathways. In conclusion, there are miRNA profiles creativecommons.org/licenses/by/ differentially expressed in OCS-treated individuals with asthma and could be used as biomarkers of 4.0/). OCS treatment. Int. J. Mol. Sci. 2023, 24, 1611. https://doi.org/10.3390/ijms24021611 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2023, 24, 1611 2 of 14 Keywords: biomarker; oral corticosteroids; miRNAs; individuals with severe asthma 1. Introduction Asthma is a heterogeneous disease of the lower airways that affects approximately 300 million people worldwide [1]. Chronic airway inflammation as well as airway damage and remodeling are hallmark features of the disease and induce bronchial hyper-reactivity and variable expiratory airflow limitation [2]. Individuals with asthma exhibit a broad spectrum of disease severity. Severe asthma, defined as that which requires treatment with high-dose inhaled corticosteroids plus a second controller and/or systemic corticos- teroids [3], affects about 5–10% of the asthmatic population, and is associated with increased mortality and morbidity, diminished quality of life, and greater health expenditure [4]. The pathophysiology of asthma is highly complex, comprising distinct phenotypes and endotypes [5]. In addition, a broad range of triggers including environmental risk factors, genetic polymorphisms, and epigenetic changes contribute to the onset of asthma, variations in phenotypes, and response to steroid treatment [6]. Severe asthma comprises two predominant inflammatory endotypes: Type 2 (T2)-high and T2-low disease [7]. T2- high asthma is more severe and difficult to treat, and many patients with this endotype require high-dose treatments and/or biological therapy for better control of the symptoms and exacerbations, and to avoid adverse reactions caused by the administration of oral corticosteroids (OCS) [8,9]. To help guide the selection of treatment in patients with severe asthma, currently avail- able biomarkers such as peripheral blood and induced sputum eosinophil count, fraction of exhaled nitric oxide (FeNO) level, and immunoglobulin-E (IgE) levels have been used [10]. However, interest in using microRNA (miRNA) profiles as biomarkers for diseases is currently on the rise [11]. These small, single-stranded, non-coding RNA molecules [12] participate in the regulation of gene expression by inhibiting protein translation [13] and are useful as biomarkers as they are stable and disease-related particles [14]. Previous studies have demonstrated that miRNAs hold great potential as asthma mediators and biomarkers and can further be of use in asthma endotyping and phenotyping, thereby mak- ing it possible to offer personalized therapies for patients [15,16]. Among them, Mirra et al. recently identified a new expression profile of circulating miRNAs including a member of the miR-181 family, related to lung inflammation, which could be used as a clinical marker of bronchial asthma and response to pharmacological treatment [17]. The aim of this study was to search for a profile of differentially expressed miRNAs between individuals with severe asthma receiving OCS treatment versus those without OCS therapy using next-generation sequencing (NGS). The miRNAs found could serve as lung-tissue or systemic biomarkers of oral corticosteroid treatment and facilitate the selection of the most appropriate management approach. 2. Results 2.1. Characteristics of the Study Population Descriptive data reflecting the demographic, inflammatory, functional, and clinical characteristics of all individuals studied are summarized in Tables 1 and 2. The subjects were distributed into two different analyses: 20 OCS-treated versus (vs.) 26 non-OCS- treated severe asthmatics (serum samples analysis) and six OCS-treated vs. seven non-OCS- treated asthmatic subjects (lung biopsy samples analysis). As shown in Table 1, regarding inflammatory parameters, the OCS-treated and non- OCS-treated severe asthmatics from which serum samples were used had significant differences in IgE levels (366.0 and 96.8 IU, p < 0.05). Additionally, as expected, we observed significant differences in treatment with systemic corticosteroids (p < 0.0001). Other characteristics (i.e., demographic, inflammatory, functional, clinical) did not present statistically significant differences. Int. J. Mol. Sci. 2023, 24, 1611 3 of 14 Moreover, comparing the treated and non-treated with OCS groups in the analysis of lung biopsy samples (Table 2), the only significant differences were detected in the percentage of monocytes (10.0% vs. 5.9%, p < 0.05) and, as planned, in the oral corticosteroid treatment (p < 0.01). Table 1. Demographic, inflammatory, functional, and clinical characteristics of the study subjects (serum sample analysis). OCS-Treated Non-OCS-Treated p-Value (n = 20) (n = 26) Age (years) † 54.5 (44.3–59.5) 53.0 (42.8–61.3) N.S. Sex (%) Female 17 (85.0%) 23 (88.5%) N.S. BMI † 26.7 (23.1–30.4) 28.7 (24.2–33.4) N.S. Smokers 1 (5.0%) 4/25 (16.0%) N.S. Smoking habit (%) Passive 2 (10.0%) 2/25 (8.0%) N.S. Ex-smokers 7 (35.0%) 9/25 (36.0%) N.S. Non-smokers 10 (50.0%) 10/25 (40.0%) N.S. Blood eosinophils (cells/µL) † 300.0 (100.0–500.0) 300.0 (200.0–525.0) N.S. Sputum eosinophils (%) † 0.0% (0.0–35.0) 3.1% (2.0–22.1) N.S. Atopy (%) 12 (60.0%) 17 (65.4%) N.S. IgE (IU) † 366.0 (110.0–690.0) 96.8 (36.6–285.8) * FEV1 /FVC (%) # 67.4% (±15.9) 73.3% (±23.5) N.S. FeNO (ppb) † 23.0 (13.5–78.3) 32.0 (13.8–62.0) N.S. Exacerbations (%) 17 (85.0%) 16 (61.5%) N.S. Severe 20 (100.0%) 26 (100.0%) N.S. Severity (%) Moderate 0 (0.0%) 0 (0.0%) N.S. Mild 0 (0.0%) 0 (0.0%) N.S. Intermittent 0 (0.0%) 0 (0.0%) N.S. ACT # 16.8 (±5.9) 17.2 (±5.5) N.S. ICS and LABA (%) 20 (100.0%) 26 (100.0%) N.S. Systemic corticosteroid (%) 20 (100.0%) 0 (0.0%) **** Results are expressed as # mean (± SD) or † median (IQR); N.S., non-significant; ****, p < 0.0001; *, p < 0.05; BMI, body mass index; FEV1 , forced expiratory volume measured during the first second; FVC, forced vital capacity; FeNO, fractional exhaled nitric oxide; ppb, parts per billion; ACT, asthma control test; ICS and LABA, inhaled corticosteroids and long-acting β2-agonists; OCS, oral corticosteroids. Table 2. Demographic, inflammatory, and clinical characteristics of the study subjects (lung biopsy sample analysis). OCS-Treated Non-OCS-Treated p-Value (n = 6) (n = 7) Age (years) † 63.0 (56.8–66.3) 34.0 (27.0–74.0) N.S. Sex (%) Female 3 (50.0%) 1 (14.3%) N.S. Smokers 1 (16.7%) 1/6 (16.7%) N.S. Smoking habit (%) Ex-smokers 3 (50.0%) 4/6 (66.7%) N.S. Non-smokers 2 (33.3%) 1/6 (16.7%) N.S. Neutrophils (%) † 61.9% (50.3–72.5) 83.6% (70.6–89.5) N.S. Lymphocytes (%) † 24.1% (16.5–35.5) 8.7% (5.9–16.5) N.S. Monocytes (%) † 10.0% (8.9–12.2) 5.9% (4.4–9.0) * Int. J. Mol. Sci. 2023, 24, 1611 4 of 14 Table 2. Cont. OCS-Treated Non-OCS-Treated p-Value (n = 6) (n = 7) Eosinophils (%) † 2.1% (0.3–3.0) 0.5% (0.1–3.1) N.S. Basophils (%) † 0.4% (0.3–0.7) 0.2% (0.1–0.5) N.S. Atopy (%) 1 (16.7%) 1 (14.3%) N.S. OCS (%) 6 (100.0%) 0 (0.0%) ** † Results are expressed as median (IQR); N.S., non-significant; **, p < 0.01; *, p < 0.05; OCS, oral corticosteroids. 2.2. MiRNA Profile in Serum Samples Analysis of the data obtained by NGS (miRNAs-seq) showed 11 differentially ex- pressed serum miRNAs (hsa-miR-148b-3p, hsa-miR-221-5p, hsa-miR-618, hsa-miR-200b-3p, hsa-miR-548I, hsa-miR-200a-3p, hsa-miR-941, hsa-miR-181d-5p, hsa-miR-769-5p, hsa-miR- 133a-3p, and hsa-miR-3614-5p) between the OCS-treated and non-OCS-treated patients with Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 5 of 14 severe asthma (adjusted p < 0.05) (Figure 1a). All of these 11 miRNAs were overexpressed in the OCS-treated subjects. Figure 1. miRNA Figure miRNA expression expressionprofile profilein inthe theserum serumsamples samplesfrom fromasthmatic asthmaticpatients. patients.(a)(a) Heatmap Heatmap of differentially expressed serum miRNAs between the OCS-treated and non-OCS-treated of differentially expressed serum miRNAs between the OCS-treated and non-OCS-treated patients patients with severe with severe asthma asthma (adjusted (adjusted pp << 0.05; 0.05; Log2 Log2 fold fold change change ≥ ≥ 1.5). 1.5). (b) (b) Principal Principal component component analysis analysis (PCA) of severe asthmatic serum samples showing the two different treatment groups (OCS-treated (PCA) of severe asthmatic serum samples showing the two different treatment groups (OCS-treated vs. non-OCS-treated severe asthmatics). (c) RT-qPCR validation of the differentially expressed se- vs. non-OCS-treated severe asthmatics). (c) RT-qPCR validation of the differentially expressed serum rum miRNAs between OCS-treated and non-OCS-treated individuals with severe asthma. 2−∆∆Ct val- −∆∆Ct values miRNAs between OCS-treated and non-OCS-treated individuals ues are represented in the graph (**, p < 0.01; *, p < 0.05). with severe asthma. 2 are represented in the graph (**, p < 0.01; *, p < 0.05). 2.3. MiRNAs Correlation and Signaling Pathways When PCA was performed using the miRNA expression values, the results revealed In order that both to establish groups somedifferentiated were clearly relation between the 1b). (Figure expression levels of these serum miR- NAs (ΔCt) and different clinical characteristics, we performed correlation analyses. We observed a significant direct correlation between FEV1/FVC and hsa-miR-148b-3p in the two groups (correlation coefficient (r) = 0.63 for the OCS-treated patients with severe asthma or correlation coefficient (r) = 0.59 for non-OCS-treated subjects with severe asthma) and hsa-miR-221-5p among the OCS-treated individuals with severe asthma (cor- Int. J. Mol. Sci. 2023, 24, 1611 5 of 14 After the miRNAs-Seq analysis, RT-qPCR validation of miRNAs was performed in a larger cohort of serum samples. Of the 11 differentially expressed miRNAs obtained by miRNAs-Seq between the OCS-treated and non-OCS-treated individuals with severe asthma, we found significantly increased levels in five of them (hsa-miR-148b-3p, hsa- miR-221-5p, hsa-miR-618, hsa-miR-941, and hsa-miR-769-5p) in the OCS-treated patients (Figure 1c). Hsa-miR-3614-5p was not detected by RT-qPCR, and the remaining miRNAs evaluated did not reach statistical significance (Figure 1c). These results confirm that serum hsa-miR-148b-3p, hsa-miR-221-5p, hsa-miR-618, hsa-miR-941, and hsa-miR-769-5p were differentially expressed miRNAs between the OCS- treated and non-OCS-treated subjects with severe asthma, suggesting that these could serve as markers of treatment. 2.3. MiRNAs Correlation and Signaling Pathways In order to establish some relation between the expression levels of these serum miRNAs (∆Ct) and different clinical characteristics, we performed correlation analyses. We observed a significant direct correlation between FEV1 /FVC and hsa-miR-148b-3p in the two groups (correlation coefficient (r) = 0.63 for the OCS-treated patients with severe asthma or correlation coefficient (r) = 0.59 for non-OCS-treated subjects with severe asthma) and hsa- miR-221-5p among the OCS-treated individuals with severe asthma (correlation coefficient (r) = 0.56) (Figure 2a). Since there is a correlation between FEV1 /FVC and hsa-miR-221-5p Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 6 of 14 in the OCS-treated severe asthmatics, it can explain the difference in treatment between the two groups, whereas, since FEV1 /FVC correlated with hsa-miR-148b-3p in both groups, we can say that, in this case, there is no explanation for the difference in treatment, but there and a0.72, was linkrespectively, with asthmaindicating pathology.that both are acceptable Furthermore, despite notas being univariate predictors. a result Fur- of statistical significance, it is worth noting the opposite trend between the groups of the OCS-treated= thermore, when both serum miRNAs were combined, the AUC was even better (AUC 0.77), and as this was the asthmatic non-OCS-treated best multivariate logistic individuals, regression in the model correlation for differentiating of hsa-miR-148b-3p OCS- with the treatedlevels, FeNO and non-OCS-treated perhaps becausesevere asthmatics OCS treatment (Figure the modified 2b).FeNO/miR-148b-3p relation. Figure Figure 2. Correlationanalyses 2. Correlation analysesand andthe theROC ROC curves curves of of differentially differentially expressed expressed serum serum miRNAs. miRNAs. (a) (a) Table showing the correlation coefficient (r) between the distinct clinical parameters and serum Table showing the correlation coefficient (r) between the distinct clinical parameters and serum miRNA miRNA expression expression levels. levels. Red color intensity indicates a higher negative correlation, whereas whereas green green color showshigher color intensity shows higherpositive positivecorrelations. correlations.Bold Bold andand underlined underlined values values werewere statistically statistically sig- nificant (p <(p0.05). significant (b) ROC < 0.05). curves (b) ROC and multivariate curves logisticlogistic and multivariate regression modelsmodels regression (continuous predic- (continuous tors) of serum predictors) hsa-miR-221-5p of serum and hsa-miR-769-5p. hsa-miR-221-5p Bold values and hsa-miR-769-5p. indicate Bold values an AUC indicate an over AUC0.70. over 0.70. When an in silico analysis was carried out to determine their involvement in the bio- logical processes with the differently expressed miRNAs, we determined that these miR- NAs are involved in the regulation of several crucial asthma pathways, which are extra- cellular matrix (ECM)–receptor interaction, fatty acid biosynthesis, steroid biosynthesis, Int. J. Mol. Sci. 2023, 24, 1611 6 of 14 ROC curves were generated, and we calculated the areas under the curve (AUC). Multivariate logistic regression models (continuous predictors) were created for better discrimination of the OCS-treated and non-OCS-treated patients with severe asthma (Sup- plementary Table S1). Hsa-miR-221-5p and hsa-miR-769-5p showed AUC values of 0.75 and 0.72, respectively, indicating that both are acceptable as univariate predictors. Furthermore, when both serum miRNAs were combined, the AUC was even better (AUC = 0.77), as this was the best multivariate logistic regression model for differentiating OCS-treated and non-OCS-treated severe asthmatics (Figure 2b). When an in silico analysis was carried out to determine their involvement in the biolog- ical processes with the differently expressed miRNAs, we determined that these miRNAs are involved in the regulation of several crucial asthma pathways, which are extracellular matrix (ECM)–receptor interaction, fatty acid biosynthesis, steroid biosynthesis, the Hippo signaling pathway, and adherens junction (Figure 3a). As TGFRB1, FOXO3, and PTEN are target genes of hsa-miR-148b-3p, and MAPK3 is the objective gene of hsa-miR-221-5p, all are involved in the signaling pathways related to asthma disease. The RT-qPCR assay was conducted in lung tissue samples from asthmatic patients and healthy subjects, revealing Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 7 of 14 that FOXO3, PTEN, and MAPK3 were significantly increased in asthmatic individuals compared to the controls (Figure 3b). Figure 3. In silico pathway analysis, genegene expression, expression, and and correlation correlation data data of of serum serum miRNA miRNA results. results. (a) Graph showing showing enrichment enrichment(as −logp value) (as–log of of p value) thethe signaling signalingpathways pathways obtained using obtained the DI- using the ANA-miRPath v3.0 DIANA-miRPath bioinformatic v3.0 bioinformatictool. (b) (b) tool. RT-qPCR RT-qPCRin the lunglung in the biopsies of target biopsies genes of target of the genes of dif- the ferentially expressed differentially serum expressed serummiRNAs miRNAs (hsa-miR-148b-3p (hsa-miR-148b-3p and hsa-miR-221-5p). and hsa-miR-221-5p). GAPDH GAPDH was used was as used a housekeeping as a housekeeping gene (*, p(*, gene < 0.05). (c) The p < 0.05). (c)relative miRNA The relative expression miRNA of hsa-miR-221-5p expression (ΔCt) corre- of hsa-miR-221-5p (∆Ct) lated directly with MAPK3 in the lung tissue samples (p < 0.05). (d) Correlation graphs of hsa-miR- correlated directly with MAPK3 in the lung tissue samples (p < 0.05). (d) Correlation graphs of hsa- 221-5p in the lung biopsy samples with blood eosinophil percentage (%) in different population miR-221-5p in the lung biopsy samples with blood eosinophil percentage (%) in different population groups (asthmatic individuals and total subjects) (p < 0.05). Dots (red and green) are individual val- groups (asthmatic ues, black individuals dashed line indicatesand errortotal barssubjects) (p < 0.05). and red/green solid Dots (red and line shows green) linear are individual regression. values, black dashed line indicates error bars and red/green solid line shows linear regression. 2.4. MiRNA Profile in Lung Samples Furthermore, to verify the association between miRNAs and the expression levels Therefore, of their validated respective serum(∆Ct), target genes miRNAs were analyzed correlation analyses in thecarried were target tissue out. We of found asthma a significant positive correlation for the total population between hsa-miR-221-5p and miR- disease. We found no statistically significant differences in the expression of these their NAs ingene, target the MAPK3 lung biopsies. For this (Spearman reason, r = 0.35) we decided (Figure 3c). to conduct miRNA-Seq in these samples from asthmatic patients in order to understand the miRNA profile in lung tissue. Here, three miRNAs were differentially expressed between the OCS-treated and non– OCS-treated asthmatics (q ≥ 8). Hsa-miR-144-3p, hsa-miR-144-5p, and hsa-miR-451a were overexpressed in the OCS-treated patients (Figure 4). We then performed RT-qPCR on asthmatic lung biopsies from a larger cohort to val- Int. J. Mol. Sci. 2023, 24, 1611 7 of 14 Finally, we examined the relation between the miRNA expression levels and eosinophil counts. We showed a statistically significant direct correlation between hsa-miR-221-5p with the percentage of eosinophils in the different population subsets (i.e., total subjects and asthmatic individuals) (Figure 3d). 2.4. MiRNA Profile in Lung Samples Therefore, validated serum miRNAs were analyzed in the target tissue of asthma disease. We found no statistically significant differences in the expression of these miRNAs in the lung biopsies. For this reason, we decided to conduct miRNA-Seq in these samples from asthmatic patients in order to understand the miRNA profile in lung tissue. Here, Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW three miRNAs were differentially expressed between the OCS-treated and non–OCS-treated8 of 14 asthmatics (q ≥ 8). Hsa-miR-144-3p, hsa-miR-144-5p, and hsa-miR-451a were overexpressed in the OCS-treated patients (Figure 4). Figure Figure 4. miRNA 4. miRNA expression expression profile profile in in thethe lung lung tissue tissue biopsies biopsies from from asthmatic asthmatic individuals. individuals. Graph Graph showing the mean NOIseq values obtained after miRNAs-Seq bioinformatic analyses (*, showing the mean NOIseq values obtained after miRNAs-Seq bioinformatic analyses (*, q ≥ 0.8). q ≥ 0.8). We then performed RT-qPCR on asthmatic lung biopsies from a larger cohort to 3. Discussion validate the miRNAs obtained by sequencing analysis. None of the three miRNAs were This article validated, drawswe however, onobserved data from a comprehensive a similar expressionstudy tendencyof the miRNAs differently in OCS-treated asthmatic expressed patients (2by− ∆∆Ct NGS for each hsa-miR: 144-3p = 6.5, 144-5p = 5.2 and 451a = 2.0), as hsa-miR- in the serum (of which 5 were validated: hsa-miR-148b-3p, obtained in 221-5p, hsa-miR-618, hsa-miR-941, and hsa-miR-769-5p) and in lung tissue (hsa-miR-144- miRNA-Seq. 3p, hsa-miR-144-5p and hsa-miR-451a) between the OCS-treated and non-OCS-treated pa- 3. Discussion tients. These miRNAs could be used as epigenetic biomarkers to distinguish individuals with severe asthmadraws This article underon OCS treatment, data although we do from a comprehensive not know study of the whether miRNAsthis miR- differently NAs alteration is due to treatment or a characteristic of patients that need expressed by NGS in the serum (of which 5 were validated: hsa-miR-148b-3p, hsa-miR-221- OCS in their asthma control. 5p, hsa-miR-618, hsa-miR-941, and hsa-miR-769-5p) and in lung tissue (hsa-miR-144-3p, Asthma is a heterogeneous hsa-miR-144-5p and hsa-miR-451a)diseasebetween comprisingthe different OCS-treated phenotypes and endotypes,pa- and non-OCS-treated and as a result, tients. These patient miRNAs stratification could be used by inflammatory endotype isto as epigenetic biomarkers a central component distinguish of individuals thewith algorithm severe used asthmato assess underand OCSmanage severe treatment, diseasewe although [18,19]. Identification do not know whether andthis appli- miR- cation NAsof biomarkers alteration used is due to to identify or treatment phenotypes and endotypes a characteristic of patientsofthat severe needasthma OCS inand their guide therapeutic asthma control. strategy is of increasing interest to clinicians, as individual approaches Asthma to disease is a heterogeneous management diseasemedicine and personalized comprising different phenotypes are imperative and endotypes, [20]. The severe asthma and as aisresult, endotype patient currently stratification defined by inflammatory by biomarkers of varying endotype is a centralblood accuracy including component and of theeosinophils, sputum algorithm used to assess periostin, FeNO,andandmanage IgE forsevere T2 asthmadisease [18,19].AsIdentification [16,21,22]. reported in the and application literature, we of biomarkers found usedsamples that serum to identify of phenotypes OCS-treatedand endotypes asthmatic of severe patients had asthma elevatedand IgEguide levelstherapeutic comparedstrategy is of increasing to the non-treated interest[23]. asthmatics to clinicians, Here, it isasworth individual approaches mentioning that to thedisease management expression and personalized of biomarkers medicine may be modified are imperative or even suppressed[20]. The severe asthma by pharmacological endotype is currently defined by biomarkers of varying accuracy including treatment (i.e., oral corticosteroids for blood eosinophils), making it advisable to examine blood and sputum eosinophils, biomarkers in combination.periostin, FeNO, and For example, weIgE for T2 that observed asthmathe [16,21,22]. combination Asof reported two serumin the literature, miRNAs usedweasfound that serum biomarkers samples of OCS-treated (hsa-miR-221-5p asthmatic resulted and hsa-miR-769-5p) patients had in a elevated better AUC than when these were analyzed individually, suggesting that the profile of these two miRNAs was greater than using them separately. Biomarkers are promising tools for the recognition and management of non-adher- ence to inhaled and oral corticosteroid therapy in asthma and can help to identify poten- Int. J. Mol. Sci. 2023, 24, 1611 8 of 14 IgE levels compared to the non-treated asthmatics [23]. Here, it is worth mentioning that the expression of biomarkers may be modified or even suppressed by pharmacological treatment (i.e., oral corticosteroids for blood eosinophils), making it advisable to examine biomarkers in combination. For example, we observed that the combination of two serum miRNAs used as biomarkers (hsa-miR-221-5p and hsa-miR-769-5p) resulted in a better AUC than when these were analyzed individually, suggesting that the profile of these two miRNAs was greater than using them separately. Biomarkers are promising tools for the recognition and management of non-adherence to inhaled and oral corticosteroid therapy in asthma and can help to identify potential responders to therapeutic options, thus enabling their use as predictors of treatment re- sponse [24]. In recent years, miRNAs have been proposed as diagnostic and prognostic biomarkers for diseases [25]. NGS is a valuable technique in these efforts and can be employed for whole genome analysis including mRNA and small RNA expression such as miRNAs [26]. Our group previously used NGS to describe 15 miRNAs differentially expressed between eosinophilic and non-eosinophilic asthmatic patients, two of which were validated and could serve as instruments to classify patients into different pheno- types/endotypes [15]. In addition, hsa-miR-144-3p was previously described by our group to present an increase in miRNA levels in corticosteroid treated subjects by RT-qPCR [27], which validates the result obtained in miRNAs-Seq from the lung biopsy samples of asth- matics. In this study, we found different profiles of miRNAs in the serum and lung tissue. Dysregulation of hsa-miR-148b-3p, hsa-miR-221-5p, hsa-miR-618, hsa-miR-941, hsa-miR- 769-5p, hsa-miR-144-3p, hsa-miR-144-5p, and hsa-miR-451a has been previously associated with different aspects of asthma pathogenesis and/or other respiratory diseases [28–32]. Nevertheless, these profiles have never been described as indicators of treatment. These miRNAs could be relevant molecules with implications for the phenotypic distinction of patients based on whether they are treated or untreated and may be indicated for use in asthma pathogenesis, since they were significantly correlated with clinical parameters. Interestingly, we found different miRNA profiles in asthmatics regarding the sample origin, which underscore the roles of these molecules as epigenetic regulators, as found in previous studies of miRNA tissue specificity [33]. Furthermore, we observed an enrichment in the ECM–receptor interaction pathway, which is widely associated with asthma due to the central role it plays in the development of airway inflammation and remodeling associated with asthma [2]. Moreover, miRNAs have been shown to affect the expression of multiple genes directly or indirectly, and for this purpose, we studied target genes related to differential miRNA expression. Here, FOXO3, PTEN, and MAPK3 exhibited increased expression in asthmatic samples, which correlated with the miRNA expression studied, which emphasizes the implication of these inflammatory genes in the disease, as previously described by others [34–36]. In addition, in this study, we observed a positive correlation between hsa-miR-221-5p and MAPK3, its target gene, instead of detecting, as usual, an inverse miRNA-target gene correlation. Given that miRNAs generally behave as silencers of their target mRNAs, a possible explanation would be that miRNA–mRNA relationships are complex, and the lung tissue samples in which they have been studied differ from each other (influence of genetic and/or environmental factors), while there may be other miRNAs involved in the modulation of a particular mRNA (MAPK3 mRNA), and there could also be interaction of other target genes of this miRNA (hsa-miR-221-5p) that could be inhibitors of MAPK3, in this case, remarking the intricacy of miRNA–mRNA post-transcriptional regulation in whole tissue. One of the main limitations of this study is the lack of the same-subject pre-treatment samples, which made it impossible to determine whether the difference in the miRNA profiles between the groups of patients treated with OCS therapy was a consequence of the treatment (reflects treatment response) or if, in contrast, the existence of differentially expressed miRNAs between the groups was a baseline characteristic of the disease and Int. J. Mol. Sci. 2023, 24, 1611 9 of 14 selected the treatment. Another limitation is the low number of lung biopsy samples available from asthmatic patients and, in fact, some of these samples were derived from individuals with other severe lung pathology, which could probably explain the reason why no significant differences were observed. Due to the limited number of analyzed lung biopsy samples available from the asthmatic patients, it is a preliminary level study in this case. Finally, it would be very interesting to evaluate the levels of protein expression by ELISA of the genes regulated by two of the altered miRNAs (hsa-miR-148b-3p and hsa-miR-221-5p) in this study. 4. Materials and Methods 4.1. Study Subjects and Sample Collection Serum samples were obtained from 46 subjects with a severe asthma diagnosis who were recruited from allergy and pulmonology units of a series of hospitals in Spain; from these, we selected samples from 20 patients undergoing treatment with OCS and 26 not receiving OCS. MiRNA-sequencing (miRNA-Seq) was performed on serum samples from six of the OCS-treated and seven non-OCS-treated patients, respectively. Of the remaining 33 samples, 14 of those obtained from OCS-treated and 19 from non-OCS-treated subjects were included for miRNA validation by means of quantitative real-time polymerase chain reaction (qPCR). Descriptive data representing the demographic, inflammatory, functional, and clinical characteristics of the study subjects were collected. All patients were participants in the MEGA project, which studies a cohort of asth- matic individuals with varying grades of severity [37]. The inclusion criteria were as follows: (i) acceptance to participate by providing signed informed consent; (ii) asthma diagnosis following the 2021 GINA criteria [38]; and (iii) age between 18 and 75 years. The definition of OCS-treated and non-OCS-treated severe asthmatic was established based on their treatment. The study was conducted in accordance with the tenets of the Declaration of Helsinki, and the protocol was approved by the participating hospital ethics committees. In addition, we analyzed the lung biopsy samples and relevant descriptive data corresponding to 13 asthmatic patient donors; these were also subdivided depending on whether the individuals were treated with OCS (OCS-treated) or not (non-OCS-treated). Furthermore, we had 20 lung biopsy samples from the control subjects, required for the gene expression analysis in lung tissue. Lung biopsy samples from these asthmatic patients and control individuals were provided by the CIBERES Pulmonary Biobank Consortium (PT13/0010/0030), a network currently formed by 12 tertiary Spanish hospitals (www.ciberes.org, accessed on 10 January 2023) detailed in the Acknowledgements section, and integrated in the Spanish National Biobanks Network. Lung biopsies were processed in accordance with the standard operating procedures, and subsequent approval was granted for processing by the local ethics and scientific committees. Serum samples were obtained by blood clotting in anticoagulant-free tubes and subse- quent centrifugation at 3000 rpm for 10 min at 4 ◦ C and stored at −80 ◦ C until use. Lung tissue samples were preserved in the RNAlater stabilization solution at −80 ◦ C until use. 4.2. Library Preparation, MiRNAs-Seq, Bioinformatic Analysis Serum RNA (including miRNAs) was extracted from 200 µL of serum using the miRNeasy Serum/Plasma Advanced Kit (Qiagen, Hilden, Germany), as indicated by the manufacturer. Lung tissue RNA was purified using the QIAzol Lysis Reagent (Qiagen, Hilden, Germany), followed by the application of the acid guanidinium thiocyanate-phenol- chloroform extraction method [39]. In all cases, the RNA enriched in miRNAs was eluted by adding 20 µL of RNase-free water. Then, small RNA (miRNA-enriched RNA) was converted to Illumina sequencing libraries using the NEXTFLEX® Small RNA-Seq Kit v3 (Bioo Scientific Corporation, Austin, TX, USA), strictly according to the manufacturer’s instructions. The size profile of the indi- vidual libraries was quantified using High Sensitivity D1000 Screentape on a 4200 TapeSta- Int. J. Mol. Sci. 2023, 24, 1611 10 of 14 tion System (both Agilent, Santa Clara, CA, USA). Quantified libraries were sequenced on an Illumina MiniSeq 550 platform (Illumina, San Diego, CA, USA) using a MiniSeq 500/550 75-Cycle High Output Kit. Bioinformatic analyses including quality control, pre-processing, and statistical analy- sis of small RNA-Seq data were carried out by the Bioinformatics Unit of IIS-Fundación Jiménez Díaz. Adaptor removal and trimming of raw reads were performed using Cu- tadapt [40] by following the instructions for the NEXTflex small RNA-Seq Kit. Adaptor- trimmed reads between 17 and 25 nt were retained and aligned to the reference genome (GRCh38 assembly) using Bowtie2 [41]. Mapping of reads to known miRNAs was per- formed with HTSeq-count2 [42] using mature miRNA annotation retrieved from the miR- Base database (miRBase v22). Raw miRNA counts across samples were normalized by sequencing depth and RNA composition using the trimmed mean of M-values (TMM) function from the NOISeq Bioconductor R package [43]. Subsequent principal component analysis (PCA) on normalized and scaled values was applied using the prcomp function from the R stats package [44]. Comparison of the normalized expression levels across groups was performed following two alternative methods for testing differential expres- sion in the sequencing data: NOISeq [43] and DESeq2 [45]. Fold-change and p-values adjusted by false discovery rate (FDR) were calculated and used to identify significant differentially expressed miRNAs. MiRNAs were considered biologically relevant if they were differentially expressed (adjusted p < 0.05) and presented a Log2 fold change ≥1.5 between two groups, or if q ≥ 0.8 in the case of NOISeq. 4.3. RT-qPCR and Pathway Enrichment Analyses For validation of the miRNA-Seq results or the expression analysis of the validated miRNAs, 4 µL of serum miRNA samples or 30 ng of lung tissue miRNA-enriched RNA were reverse transcribed to cDNA using the miRCURY LNA RT Kit (Qiagen), following the manufacturer’s protocol. The synthetic miRNAs SP6 and cel-miR-39-3p were used to control for correct reverse transcription. The cDNA obtained was stored at −20 ◦ C until use. Then, miRNA expression was evaluated by qPCR using the miRCURY LNA SYBR Green PCR Kit (Qiagen), as indicated in the instructions. For this purpose, we used 3 µL of cDNA from the serum or lung tissue miRNAs diluted 1:60 or 1:30, respectively, in RNase-free water. Based on the results of the miRNA-Seq, the probes (Qiagen) used for the validation analysis of miRNAs in the serum and lung tissue were the following: hsa-miR-148b-3p, hsa-miR-221-5p, hsa-miR-618, hsa-miR-200b-3p, hsa-miR-548I, hsa-miR- 200a-3p, hsa-miR-941, hsa-miR-181d-5p, hsa-miR-769-5p, hsa-miR-133a-3p, hsa-miR-451a, hsa-miR-144-3p, and hsa-miR-144-5p. Additionally, hsa-miR-103a-3p, hsa-miR-191-5p, SP6, cel-miR-39-3p, and U6 (Qiagen) were selected as housekeeping miRNAs. All samples were run in triplicate, and reactions were performed in a Light Cycler® 96 thermocycler (Roche, Basel, Switzerland). Cycle threshold (Ct) values were analyzed with LightCycler® 96 SW 1.1 (Roche) software and miRNA expression was calculated by normalizing to the endogenous miRNA controls by applying the 2−∆Ct method [46], where ∆Ct = CtmiRNA − Cthousekeeping miRNAs . The relative quantification of differences in expression (RQ = 2−∆∆Ct ; where ∆∆Ct = ∆CtOCS-treated − ∆Ctnon-OCS-treated ) was carried out by the ∆∆Ct method [46]. For gene expression analysis in lung tissue, 500 ng of RNA quantified by a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA), followed by qPCR according to the manufacturer’s guidelines on a StepOne Real-Time PCR System (Applied Biosystems). TaqManTM gene expression probes were purchased for TGFBR1, FOXO3, PTEN, MAPK3, and GAPDH using TaqManTM Gene Expression MasterMix (Applied Biosystems). Gene expression was calculated by normalizing to the endogenous gene GAPDH control by applying the 2−∆Ct method as previously reported (RQ = 2−∆∆Ct ; where ∆∆Ct = ∆Ctasthmatics − ∆Cthealthy subjects ) [46]. In order to identify target genes linked to asthmatic pathology of hsa-miR-148b-3p, hsa-miR-221-5p, hsa-miR-618, hsa-miR-941, and hsa-miR-769-5p, differentially expressed Int. J. Mol. Sci. 2023, 24, 1611 11 of 14 miRNAs in the serum between the OCS-treated and non-OCS-treated patients with severe asthma, pathway enrichment analysis of dysregulated miRNAs was performed using the DIANA-miRPath v3.0 bioinformatic online resource [47]. Relevant pathways for asthma disease were represented when a p-value < 0.05, and the related genes were analyzed by RT-PCR, as previously mentioned. 4.4. Statistical Analysis Statistical analyses and graphs were created with GraphPad Prism® v6-8.0 (GraphPad Software Inc., San Diego, CA, USA) and R software® v4.1.0 [44] (R Foundation for Statistical Computing, Vienna, Austria). Results are shown as the mean (standard deviation, SD) or median (interquartile range, IQR) values. For all statistical analyses, differences showing p < 0.05 were considered significant. Normality was analyzed by means of the Shapiro–Wilk test. For continuous variables, comparisons of normally distributed data between non-paired groups were performed via an unpaired t test (equal SD) and t test with Welch’s correction (different SD), and non-normally distributed data and non-paired groups were compared by the Mann–Whitney test. Additionally, correlations between the miRNA expression levels (∆Ct) and some clinical parameters (quantitative variables) were estimated by Spearman (non-normally distributed data) or Pearson (normally distributed data) correlation; the Fisher exact test was applied to a 2 × 2 contingency table to test the null hypothesis of the independence of groups and some clinical characteristics (qualitative variables). Finally, the expression profile (∆Ct) of each differentially expressed miRNA was used to create receiver operator characteristic (ROC) curves, and logistic regression models were developed to evaluate the performance of miRNA as biomarkers; an area under the curve (AUC) of 0.7 indicated an acceptable biomarker. 5. Conclusions In summary, we describe the significant differences in the expression of eight miRNAs, hsa-miR-148b-3p, hsa-miR-221-5p, hsa-miR-618, hsa-miR-941, hsa-miR-769-5p, hsa-miR- 144-3p, hsa-miR-144-5p, and hsa-miR-451a (the first five in serum and the last three in lung tissue). These miRNAs could be used as biomarkers of oral corticosteroid treatment, allowing for the differentiation of patients treated with OCS from the patients not treated with OCS. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ijms24021611/s1. Author Contributions: V.d.P. conceived the manuscript and designed the study. M.G.-M., C.L.-S., J.M.R.-M. and M.Á.L. performed the experiments and data analysis. M.G.-M. and C.L.-S. edited and wrote the manuscript. G.N.-M., L.d.l.F., P.M. and I.M.-F. performed the bioinformatics and statistical data analysis. J.S., M.V.-M., S.Q., M.L.C., F.J.G.-B., E.A., I.B., A.V., X.M., M.J.C., C.M.-R., V.P. and J.M.O. recruited the patients and obtained the samples and clinical data. All authors contributed to the article and approved the submitted version. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by ISCIII—Instituto de Salud Carlos III, FIS (Fondo de Investi- gación Sanitaria—Spanish Health Research Fund) grants PI18/00167, PI21/00896, and FI19/00067; Ciber de Enfermedades Respiratorias (CIBERES); RTC-2017-6501-1 (Ministerio de Ciencia, Innovación y Universidades), a Carlos III Institute of Health Initiative; and FEDER funds (Fondo Europeo de Desarrollo Regional). Institutional Review Board Statement: The study protocol was approved by the Ethics Committee of each different hospital, and the study was conducted in accordance with the principles set forth in the Declaration of Helsinki. Int. J. Mol. Sci. 2023, 24, 1611 12 of 14 Informed Consent Statement: Signed informed consent was obtained from all patients. The study was conducted in accordance with the tenets of the Declaration of Helsinki, and the protocol was approved by the participating hospital ethics committees. Data Availability Statement: The data that support the findings of this study are available from the corresponding author, V.d.P., upon reasonable request. Acknowledgments: We wish to particularly acknowledge the patients and the CIBERES Pulmonary Biobank Consortium (PT13/0010/0030) on behalf of Hospital Universitario 12 de Octubre, Hospital del Mar-IMIM, Hospital Universitario de Getafe, Consorcio Universitario Hospital General de Va- lencia, Fundación Jiménez-Díaz, Hospital Clínic de Barcelona, Hospital Universitario de Bellvitge, Hospital Universitario Joan XXIII, Hospital Universitario Germans Trias i Pujol, Hospital Universi- tario Virgen del Rocío, Hospital Vall d’Hebron, and Hospital Universitario Son Espases, integrated in the Spanish National Biobanks Network, for their collaboration. The authors also acknowledge Oliver Shaw for the editorial support. Conflicts of Interest: Authors declare that the research was conducted in the absence of any commer- cial or financial relationships that could be construed as potential conflict of interest. References 1. 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