MiR-26b-5p and its association with inflammatory markers in infants and toddlers with bronchiolitis and respiratory symptoms
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Lanlan Li
,
Yanrui Yan
,
Shuge Wang
and
Wenlong Yi
Abstract
Objectives
The aim of this study was to investigate the impact of miR-26b-5p on diagnosis of bronchiolitis as well as its relationship with inflammatory factors.
Methods
This study conducts a retrospective study of infants and toddlers with bronchiolitis, coughing and wheezing as the main symptoms (RSV-positive and RSV-negative). The qRT-PCR was used to examine miR-26b-5p expression levels. Diagnostic accuracy of miR-26b-5p in infant and toddler respiratory diseases was assessed by ROC curve. Pearson correlation analysis was applied to demonstrate the relationship between miR-26b-5p and clinical severity markers, along with immune-related genes such as PTGS2 and TRAF3. RSV-infected HBECs were used to validate the effect of miR-26b-5p on inflammatory factors. IL-6, IL-10 and TNF-α were measured by ELISA kits.
Results
MiR-26b-5p expression was reduced in infant and toddler respiratory diseases including bronchiolitis, coughing and wheezing. The ROC curve indicated that miR-26b-5p had a high diagnostic accuracy for differentiating respiratory diseases from healthy infants and toddlers. Notably, miR-26b-5p returned to baseline in infants and toddlers who had recovered from bronchiolitis. Pearson correlation analysis revealed a negative relationship of miR-26b-5p with PTGS2 and TRAF3. RSV infection of HBECs reduced miR-26b-5p expression, and transfection with miR-26b-5p mimic reversed this trend. Furthermore, IL-6 and TNF-α levels were significantly elevated and IL-10 levels were reduced in RSV-infected HBECs, which was reversed by overexpression of miR-26b-5p.
Conclusions
MiR-26b-5p may be involved in disease progression through modulation of inflammatory responses and may be a diagnostic biomarker for infant and toddler respiratory diseases.
Introduction
The underdeveloped respiratory system of infants and toddlers is characterized by narrowness of respiratory tract, insufficient secretion of mucus glands, and poor cilia movement, features that predispose them to respiratory infections [1]. Infant and toddler respiratory diseases are the leading cause of morbidity and mortality among children under 5 years old worldwide [2]. Bronchiolitis is a frequent occurrence of lower respiratory tract infection, mainly caused by pathogens such as respiratory syncytial virus (RSV) [3]. The disease is recognized by airway inflammation, increased mucus secretion and bronchial obstruction, with clinical symptoms such as wheezing, coughing and dyspnea, which in severe cases may lead to respiratory failure or even death [4]. The data in the literature indicates an increased rate of lower respiratory infections (bronchiolitis and pneumonia) and wheezing in short-and mid-term follow-ups, and treatment with palivizumab reduced its occurrence [5], 6]. However, the data for asthma remains inconclusive. While RSV infection in infancy has been linked to asthma development in later childhood, there is conflicted evidence, and the causality of this relationship is still debated as the underlying mechanism is not known [7], 8]. Therefore, in-depth exploration of the pathogenesis of infant and toddler respiratory diseases and searching for new biomarkers have important clinical significance.
MicroRNAs (miRNAs) play a key role in gene expression regulation and immune response [9]. It regulates disease processes by affecting the stability of mRNAs and inhibiting their transcription, translation or degradation [10]. Multiple miRNAs were detected to take part in infant respiratory disease regulation. For example, miR-146a-5p has reduced expression in infant respiratory disease and exerts a regulatory function by mediating immune modulation [11]. MiR-34b/c regulated mucus secretion in RSV-infected airway epithelial cells by targeting FGFR1 [12]. MiR-26b-5p has been reported to show downregulation in inflammatory diseases such as asthma and chronic obstructive pulmonary disease [13]. In addition, the downregulation of miR-26b-5p expression was discovered in the nasal airways of infants hospitalized for RSV-infected bronchiolitis [14]. Studies have revealed that the pathological process of bronchiolitis was closely related to the release of inflammatory mediators such as IL-6, IL-10, TNF-α [15]. Notably, miRNAs may be key molecules linking viral infection to the host immune response by targeting and regulating the expression of inflammatory factors or signaling pathways [16]. It has been suggested that overexpression of miR-140-5p inhibited the inflammatory response and reduced IL-1β, IL-6 and IL-8 levels, which in turn regulated RSV infection disease [17]. Jiang et al. demonstrated that miR-26b-5p alleviated acute soft tissue injury by inhibiting the inflammatory factors TNF-α, IL-6 and IL-1β through the regulation of COX2 [18]. However, the immune and inflammatory responses of miR-26b-5p in bronchiolitis have not yet been investigated.
In this study, we assessed the role played of miR-26b-5p in infant and toddler respiratory disease by comparing its expression in diseased and healthy infants and toddlers. Moreover, we analyzed the expression of miR-26b-5p in relation to immune-related genes and the regulation of inflammatory factors by in vitro experiments. The aim is to provide a novel biomarker for infant and toddler respiratory disease diagnosis, as well as a new theoretical basis for development and regulation of this disease.
Materials and methods
Sample collection
This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethical Research Committee of Tianyou Hospital Affiliated to Wuhan University of Science and Technology and included infants and toddlers whose families were notified and signed an informed consent.
This research is a retrospective study. The sample size was calculated by setting a two-sided test α=0.05 and taking 1-β=0.80 to ensure the reliability of detecting differences between groups. It also ensured that the difference δ≥15 % for the primary indicator and δ≥20 % for the RSV-positive and RSV-negative subgroups. On this basis, the minimum sample size for a single group was calculated to be 76 cases, and the RSV-positive and RSV-negative subgroups each required 43 cases. Therefore, we chose 100 infants and toddlers with bronchiolitis and 100 infants and toddlers (50 RSV-positive and 50 RSV-negative) with coughing and wheezing as the main symptoms admitted to our hospital from 2021 to 2024. Besides, a total of 100 healthy infants and toddlers of the same age and period from the child health clinic were selected as a control group. Inclusion criteria for bronchiolitis infants and toddlers consisted of age at 1–24 months, clinical presentation of first wheezing with cough, fever, shortness of breath, lung rales, wet rales, no secondary bacterial infections, and no use of systemic glucocorticoids. Exclusion criteria included combination of chronic lung disease, congenital heart disease, presence of immunodeficiency or inherited metabolic disorders, use of immunomodulators or antiviral drugs within two weeks. Inclusion criteria for the cough and wheezing group were age at 1–24 months, clinical diagnosis with symptoms of dyspnea, wheezing, hypoxia, apnea or respiratory distress, and rales or prolonged expiratory phase heard on lung auscultation. Exclusion criteria included congenital heart disease and bronchopulmonary dysplasia, the presence of immunodeficiencies and inherited metabolic disorders, comorbid bacterial pneumonia or tuberculosis, and the use of systemic glucocorticoids, antivirals, or immunomodulators within two weeks. Infants in the control group were aged 1–24 months, previously healthy, without a history of wheezing diseases such as bronchiolitis, and without a familial history of atopy.
Basic information about the research subjects was collected by searching electronic medical records, including age, gender, prematurity, hospital stay, and whether they were RSV infected.
Peripheral blood and nasopharyngeal swabs collection
The infants and toddlers were immobilized on all four limbs, keeping the puncture side extended, and 3 mL of blood was collected from the elbow vein, femoral vein, or scalp vein in a vacuum blood collection tube. After collection, the blood was allowed to stand at 4 °C for 1 h, then centrifuged at 3,000 rpm/min for 5 min. Subsequently, the upper serum layer was removed to a new freezing tube and placed in a −80 °C refrigerator (Thermo, USA) for storage.
A nasal discharge or crust is cleared to ensure that the swab can pass through the lower nasal passages. The swab was inserted vertically along the lower nasal passage to the posterior wall of the nasopharynx, rotated 3–5 times in response to resistance, and held for 15 s to absorb secretions. After collecting, the swab stalk broke along the crease and placed in a centrifuge tube with 3 mL PBS buffer, mixed upside down and stored at −80 °C in a refrigerator for later use.
Cell culture
Normal primary human bronchial epithelial cells (HBECs) were purchased and cultured in DMEM medium (Gibco, USA). The medium contained 10 % fetal bovine serum (HyClone, USA) and 1 % penicillin-streptomycin (Gibco, USA) and was cultured in an incubator (Thermo, USA) at 5 % CO2 and 37 °C. The cells were left to culture until the logarithmic growth phase for subsequent assays.
RSV infection and cell transfection
Purified RSV (mol=3) was incubated with HBECs and adsorbed in an incubator with 5 % CO2 at 37 °C for 90 min. RSV virus infection was performed by slow shaking every 10 min to homogenize virus adsorption.
MiR-26b-5p mimic or mimic NC was transfected into HBECs and RSV-infected HBECs using Lipofectamine 2000 (Invitrogen, USA). Cells were obtained after transfection for 48 h and used for further experiments.
RNA extraction and qRT-PCR
Total RNA was extracted by adding Trizol reagent (Invitrogen, USA) to serum or cultured cells. RNA was reversed to cDNA according to RevertAid First Strand cDNA Synthesis Kit (Invitrogen, USA) instructions. The qRT-PCR reaction was followed according to the steps of SYBR Green PCR Master Mix Kit (Invitrogen, USA). The gene expression was calculated using the ΔΔCt method with U6 as a housekeeping gene for miR-26b-5p and GAPDH for PTGS2 and TRAF3. The relative quantified values (2−ΔΔCt) were log2 transformed to an approximate normal distribution for parametric statistical analysis. Three independent replicates were performed for each sample. Nasopharyngeal swab samples were extracted for RNA using BIOG RNA kit (BAIDAI, China) and then analyzed by qRT-PCR. The PCR primers were as follows: miR-26b-5p forward, 5′-GCCGAGUUCAAGUAAUUCA-3′ and reverse, 5′-CTCAACTGGTGTCGTGGA-3′; PTGS2 forward, 5′-GTTCCACCCGCAGTACAGAA-3′ and reverse, 5′-AGGGCTTCAGCATAAAGCGT-3′; TRAF3 forward, 5′-ACCGCGAGAACTCCTCTTTC-3′ and reverse, 5′-TCAGGGACAAAAACTGGCGT-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′; GAPDH forward, 5′-CATCAACGGGAAGCCCATC-3′ and reverse, 5′-CTCGTGGTTCACACCCATC-3′.
IL-6, IL-10 and TNF-α detection
RSV-infected HBECs cells were collected and centrifuged at 3,000 rpm for 10 min to remove cell debris. IL-6 (NO:430501, BioLegend, USA), IL-10 (NO:430601, BioLegend, USA), and TNF-α (NO:430201, BioLegend, USA) levels were measured under the instructions of ELISA kits.
Statistical analysis
SPSS 27.0 and GraphPad Prism 9.0 software were applied for statistical analyses and graphing. The Kolmogorov-Smirnov test was used to determine the distribution of continuous variables, and data were expressed as mean ± SD. Comparisons between groups were made using Student’s t test, and multiple comparisons were made using one-way ANOVA. The accuracy of miR-26b-5p in diagnosing respiratory diseases occurring in infants and toddlers was analyzed using ROC curves. Pearson correlation analysis was applied to assess the relationship between miR-26b-5p and immune-related genes. The test used p<0.05 indicates a statistically significant difference.
Results
Basic information on research subjects
According to the inclusion criteria of each group, a total of 300 study subjects were enrolled in our research, including 100 cases in control group with a mean age of 13.78 ± 6.14 months, 100 cases in bronchiolitis group with a mean age of 11.31 ± 6.83 months, 50 cases in RSV-positive coughing and wheezing group with a mean age of 11.66 ± 6.58 months, and 50 cases in RSV-negative coughing and wheezing with a mean age of 12.60 ± 6.40 months. It is well known that RSV is the main cause of bronchiolitis. In our research subjects, 77 % of the infants and toddlers in the bronchiolitis group were infected with RSV. Additional details of the study population are given in Table 1.
Basic information on research subjects.
| Control (n=100) | Bronchiolitis (n=100) | Coughing and wheezing, RSV-positive (n=50) | Coughing and wheezing, RSV-negative (n=50) | |
|---|---|---|---|---|
| Age, month | 13.78 ± 6.14 | 11.31 ± 6.83 | 11.66 ± 6.58 | 12.60 ± 6.40 |
| Gender (male/female) | 56/44 | 58/42 | 29/21 | 24/26 |
| Prematurity | NA | 4 | 2 | 1 |
| Hospital stay | NA | 5.04 ± 1.10 | 2.64 ± 0.96 | 2.66 ± 0.82 |
| Temperature>37.9 °C | NA | 15 | 22 | 17 |
| Hypoxia (SatO2<95 %) | NA | 67 | 38 | 26 |
| Duration of oxygen therapy, hours/day | NA | 11.96 ± 3.29 | 10.10 ± 2.79 | 9.08 ± 2.19 |
| RSV | NA | 77 | 50 | NA |
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Data are shown as mean ± SD. SatO2, oxygen saturation; RSV, respiratory syncytial virus; NA, not available.
MiR-26b-5p expression was reduced in infant and toddler respiratory disease
The qRT-PCR was used to examine peripheral blood samples, and the results showed that miR-26b-5p expression was reduced in infants and toddlers with respiratory diseases in comparison with healthy (p<0.001, Figure 1A). Grouped according to disease episodes, the results demonstrated that miR-26b-5p expression was lower in bronchiolitis and coughing and wheezing groups than in the control group, but there was little difference between RSV-positive and RSV-negative within the groups (p<0.001, Figure 1B). The receiver operating characteristic (ROC) curve indicated that miR-26b-5p had the ability to significantly discriminate between healthy and respiratory disease infants and toddlers, with a sensitivity of 82 % and a specificity of 83 % (AUC=0.895, p<0.001, Figure 1C). In addition, we analyzed the association between miR-26b-5p and severity markers in infants and toddlers with RSV-positive bronchiolitis. As shown in Table 2, miR-26b-5p was significantly negatively correlated with hospital stay (r= −0.567, p<0.001), temperature (r= −0.571, p<0.001), and duration of oxygen therapy (r= −0.725, p<0.001), and positively correlated with hypoxia (r= 0.628, p<0.001).
miR-26b-5p expression in infant and toddler respiratory disease. (A) miR-26b-5p expression was reduced in infant and toddler respiratory disease. p value, result of Student’s t test. (B) miR-26b-5p expression was lower in bronchiolitis and coughing and wheezing groups. p value, result of one-way ANOVA. (C) ROC curve of miR-26b-5p for diagnosis of infant and toddler respiratory diseases. Date are shown as mean ± SD, ***p<0.001.
Correlation between miR-26b-5p and severity markers in RSV-infected bronchiolitis infants and toddlers.
| Correlation with miR-26b-5p, r | p-Value | |
|---|---|---|
| Hospital stay | −0.567 | <0.001 |
| Temperature>37.9 °C | −0.571 | <0.001 |
| Hypoxia (SatO2<95 %) | 0.628 | <0.001 |
| Duration of oxygen therapy, hours/day | −0.725 | <0.001 |
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Data are shown as mean ± SD. SatO2, oxygen saturation. r and p value, result of Pearson correlation analysis. p<0.05 indicates a statistically significant difference.
Normalization of miR-26b-5p levels in cured infants and toddlers
To determine which changes in miR-26b-5p levels occur in infants and toddlers with respiratory disease after remission, we analyzed infants and toddlers with post bronchiolitis and ongoing bronchiolitis. The expression of miR-26b-5p was measured in peripheral blood and nasopharyngeal swabs of infants and toddlers with bronchiolitis, respectively. It was found that miR-26b-5p remained decreased in peripheral blood and nasopharyngeal swabs in infants and toddlers with ongoing bronchiolitis, whereas miR-26b-5p levels returned to normal in both peripheral blood and nasopharyngeal swabs in post bronchiolitis (p<0.001, Figure 2A and B).
Normalization of miR-26b-5p levels in cured infants and toddlers. (A, B) miR-26b-5p remained decreased in peripheral blood and nasopharyngeal swabs in infants and toddlers with ongoing bronchiolitis, whereas miR-26b-5p levels returned to normal in post bronchiolitis. p value, result of one-way ANOVA. Date are shown as mean ± SD,***p<0.001; ns, non-significant.
MiR-26b-5p was negatively correlated with PTGS2 and TRAF3.
To investigate the association between miR-26b-5p and immune dysregulation in bronchiolitis infants and toddlers, we explored the correlation between miR-26b-5p expression levels and the immune-related genes PTGS2 and TRAF3 by testing peripheral blood. Pearson correlation analysis as illustrated in Figure 3A and B, miR-26b-5p expression was negatively correlated with both PTGS2 and TRAF3 levels (r PTGS2 = −0.715, r TRAF3 = −0.459, p<0.001).
Correlation of miR-26b-5p with immune-related genes. (A, B) miR-26b-5p was negatively correlated with PTGS2 and TRAF3. r and p value, result of Pearson correlation analysis.
MiR-26b-5p upregulation reversed RSV-induced elevated levels of IL-6, IL-10 and TNF-α
To examine the impact of miR-26b-5p overexpression on inflammatory factors in RSV-infected cells, we carried out by transfecting miR-26b-5p into HBECs. Transfection with miR-26b-5p mimic into normal HBECs resulted in a remarkable elevation of miR-26b-5p levels, proving that miR-26b-5p vector construction was successful (p<0.001, Figure 4A). Further transfection with miR-26b-5p mimic into RSV-infected HBECs found that miR-26b-5p expression was reduced after cells were infected with RSV, whereas miR-26b-5p mimic significantly reversed this trend (p<0.001, Figure 4B). Furthermore, changes in IL-6, IL-10 and TNF-α were detected, which showed that IL-6 and TNF-α levels were increased markedly and IL-10 level was decreased after RSV infection in HBECs. However, overexpression of miR-26b-5p reversed their levels (p<0.001, Figure 4C–E).
Effect of miR-26b-5p overexpression on RSV-infected HBECs. (A) miR-26b-5p expression is elevated after transfection with miR-26b-5p mimic in HBECs. (B) miR-26b-5p expression was reduced after RSV infection of HBECs, and transfection of miR-26b-5p mimic reversed this trend. (C–E) RSV infection of HBECs resulted in elevated levels of IL-6 and TNF-α, and reduced levels of IL-10, which was reversed by transfection with miR-26b-5p mimic. p value, result of one-way ANOVA. Date are shown as mean ± SD, ***p<0.001.
Discussion
Respiratory diseases are a group of illnesses that are prevalent and have a great impact on infants and toddlers, including pneumonia, bronchiolitis, influenza, and wheezing episodes [19]. Bronchiolitis is one of the most common respiratory diseases in infancy. The present study revealed significant downregulation of miR-26b-5p in infants and toddlers with bronchiolitis and correlated with IL-6, IL-10, TNF-α inflammatory markers and immune dysregulation genes PTGS2 and TRAF3, suggesting that miR-26b-5p may play a key role in respiratory diseases in infants and toddlers.
The means of diagnosing diseases by detecting the expression of miRNAs is gaining more and more attention in medicine [20]. In infant and toddler respiratory disease, miRNAs were identified to be involved in its regulation. For example, miR-146a, miR-145 were involved in the progression of pneumonia in RSV-infected children and had a diagnostic value [21]. Low miR-26b-5p expression levels identified in research on asthma and chronic obstructive pulmonary disease may be a potential biomarker [13]. In our study, we identified lower miR-26b-5p expression levels in infant and toddler respiratory diseases, consistent with previously examined expression trends. ROC curve analysis of miR-26b-5p showed high sensitivity and specificity for diagnosis of infant and toddler respiratory diseases. These results suggested that miR-26b-5p may be a biomarker for diagnosis of infant and toddler respiratory diseases. In contrast to earlier studies showing upregulation of miR-26b in peripheral blood mononuclear cells, this study showed that miR-26b-5p was inhibited in RSV-infected HBECs [22]. This difference may arise from specific regulation by cell type or disease stage. Therefore, the dynamic expression of miR-26b-5p in bronchiolitis requires further elucidation.
Impairment of respiratory barrier function may enhance the activity of cellular pro-inflammatory factors [23]. Downregulation of miR-26b-5p may impair its inhibitory effect on pro-inflammatory pathways. IL-6 and TNF-α, as typical pro-inflammatory factors, are released in large quantities during RSV infestation of infants [24]. IL-10 is a key anti-inflammatory cytokine, and Laubreton et al. revealed that regulatory B lymphocytes, enriched in neonatal lungs, interact with alveolar macrophages to modulate RSV replication through the inhibition effects of IL-10 on the IFN-Ⅰ response [25], 26]. Previous studies have demonstrated that low miR-26b-5p expression promotes higher IL-6 and TNF-α levels on fibroblast-like synovial cells in rheumatoid arthritis [27]. Consistent with previous findings, our study confirmed the regulation of inflammatory responses by miR-26b-5p in RSV-infected HBECs. This suggested that miR-26b-5p may alleviate inflammatory response in infant and toddler respiratory diseases.
The immune system has the roles of immune defense, immune self-stabilization and immune surveillance, as well as being an important system for the body to carry out immune response functions [28]. Immune dysregulation is often triggered when respiratory diseases occur in infants and toddlers [29]. The targeting relationship between miR-26b-5p and PTGS2 and TRAF3 was retrieved from the miRDB database. In exploring Houttuynia cordata Thunb’s study of RSV disease, Du et al. identified a central role for PTGS2 in the antiviral process [30]. Moreover, TRAF3 levels were regulated by NS1 and NS2 proteins by different mechanisms in RSV infection [31]. Our results showed that PTGS2 and TRAF3 were negatively correlated with miR-26b-5p expression. This demonstrated that miR-26b-5p may participate in immune dysregulation triggered by infant and toddler respiratory diseases.
This study was only a preliminary investigation in HBECs, and further animal model validation is required for the function played by miR-26b-5p in infant and toddler respiratory diseases. In addition, miR-26b-5p may be differentially regulated by different pathogens or disease stages, requiring pathogen-specific analyses and dynamic expression studies.
In summary, miR-26b-5p was significantly reduced in infants and toddlers with bronchiolitis, coughing and wheezing and its levels correlated with inflammatory factors, suggesting that miR-26b-5p may be involved in disease progression through modulation of inflammatory responses and may be a diagnostic biomarker.
Funding source: Youth Talent Project of Hubei Provincial Department of Education
Award Identifier / Grant number: Q20201103
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Research ethics: This study was approved by the Ethical Research Committee of Tianyou Hospital Affiliated to Wuhan University of Science and Technology.
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Informed consent: Included infants and toddlers whose families were notified and signed an informed consent.
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Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission. All authors contributed to the study conception and design. Study concept and design: L.L. L., and W.L. Y.; analysis and interpretation of data: L.L. L., and Y.R. Y.; drafting of the manuscript: L.L. L.; critical revision of the manuscript for important intellectual content: L.L. L., Y.R. Y., and W.L. Y.; statistical analysis: L.L. L., and S.G. W.
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Use of Large Language Models, AI and Machine Learning Tools: None declared.
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Conflict of interest: The authors state no conflict of interest.
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Research funding: This study was funded by Youth Talent Project of Hubei Provincial Department of Education (Q20201103).
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Data availability: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
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