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J Investig Allergol Clin Immunol 2019; Vol. 29(5): 378-398

© 2019 Esmon Publicidad

written informed consent to participate. The study was

conducted following the principles of the Declaration of

Helsinki and approved by Fundación Jiménez Díaz Ethics

Committee. All selected patients had a confirmed diagnosis

of asthma with >12% improvement in FEV


15 minutes

after inhaling salbutamol (400 μg) or methacholine airway

hyperresponsiveness (PC


methacholine <16 mg/mL) [9].

They also had moderate persistent asthma and were being

treated with a combination of inhaled corticosteroids/long-

acting ß-agonists at medium doses (400 µg of budesonide and

12 µg of formoterol fumarate dihydrate daily or equivalent).

No change was made in the treatment received for asthma

during the study period, ie, from baseline to the follow-up

visits. Serum was obtained by centrifugation and stored at

–80ºC before analysis for no more than 2 years.

Serum miRNAs were extracted using the miRCURY

RNA Isolation Kit-Biofluids (Qiagen) and retrotranscribed

to cDNA using the Universal cDNA Synthesis kit II (Qiagen)

following the manufacturer’s instructions. Synthetic spike-ins

were added during the RNA extraction (Sp2, Sp4, and Sp5)

and reverse transcription (Sp6) processes to ensure appropriate

extraction and cDNA synthesis. miRNA expression was

evaluated using quantitative polymerase chain reaction as

previously described [10] at baseline and 6-12 months later

at follow-up visits.

The miRNAs analyzed were miR-320-a, miR-144-5p,

miR-1246, miR-21-5p, and miR-185-5p. These miRNAs

were selected because we previously found that their profile in

eosinophils can be used as a serum biomarker of asthma [10].

MiR-191-5p was measured as the endogenous control, and

Sp2, Sp4, Sp5, and Sp6 were measured to ensure correct

extraction and reverse transcription.

The statistical analysis was carried out using the GraphPad

Instat program. The


test was performed for normally

distributed samples (those meeting a Gaussian distribution)

and the Mann-Whitney test for non-normally distributed

samples. Paired tests were performed to compare baseline

data with follow-up data.

Asthma was stable over time in terms of the mean (SD)

Asthma Control Test score (21.1 [3.7] vs 20.8 [3.1]) and lung

function (FEV


%, 97.7 [12.9] vs 97.5 [13.9]). In addition

to the stable clinical parameters, no statistically significant

differences were found between the results obtained in

asthmatics at baseline and follow-up visits for any of the

Stability of Asthma Control Implies No Changes in

microRNAs Expression

Rial MJ


*, Rodrigo-Muñoz JM


*, Sastre B


, Sastre J


, del

Pozo V



Department of Allergy, Hospital Universitario Fundación

Jimenez Diaz, Instituto de Investigación Sanitaria Fundacion

Jimenez Diaz (IIS-FJD, UAM), Madrid, Spain


Department of Immunology, Instituto de Investigación Sanitaria

Hospital Universitario Fundacion Jimenez Diaz. (IIS-FJD, UAM),

Madrid, Spain


CIBER de Enfermedades Respiratorias (CIBERES), Instituto de

Salud Carlos III, Spain


These authors contributed equally to the manuscript.

J Investig Allergol Clin Immunol 2019; Vol. 29(5): 388-389

doi: 10.18176/jiaci.0410

Key words:

Asthma. miRNA. Biomarkers. Eosinophils. Exosomes.

Palabras clave:

Asma. miRNA. Biomarcadores. Eosinófilos. Exosomas.

Asthma is a chronic disease that affects 4.3% of the

population worldwide [1]. Pulmonary function tests and

bronchial provocation tests are still the gold standard in

diagnosing and assessing the severity of respiratory diseases;

however, they are not able to differentiate between the clinical

phenotypes responsible for specific manifestations. An ideal

biomarker must be measurable with minimal invasiveness,

be specific and sensitive, and be able to be detected quickly

and accurately. In this context, microRNAs (miRNAs)

present in body fluids have been reported to meet several

of these criteria and are used as diagnostic markers in

many areas [2-4]. Eosinophils, which play a key role in the

pathogenesis of asthma, have the ability to secrete exosomes.

These structures contain miRNAs, which are single-stranded

RNA sequences (around 19-22 nucleotides) that do not code

for proteins with crucial functions in the development and

maintenance of the pathogenic mechanisms of asthma, but

may instead be implicated in the pathophysiology of asthma

by regulating the translation of proteins related to asthma

processes [5]. miRNAs can be encapsulated in exosomes or

bound to proteins in biofluids; in both cases, they are highly

resistant to degradation by RNases [6,7].

Released exosomes and the miRNAs inside them have

been found in serum. However, neither the precise role of

miRNAs in asthma nor their stability in the same patient over

time has been fully defined [3-5].

In order to establish whether expression of miRNA in

clinically stable asthma patients remains steady over time, we

selected 20 asthmatic patients from a national cohort (MEGA

project) [8]. These patients were recruited randomly in the

Allergy Department of Fundación Jiménez Díaz University

Hospital, Madrid, Spain. Clinical and epidemiological

characteristics are shown in the Supplementary Material.

Patients received all necessary information and gave their


Expression of miRNAs by qPCR at baseline and at follow-up visits.

Relative expression (2


) is shown as mean (SD).









miR-320a miR-185-5p



Relative miRNA Expression (2