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Pulmonary microRNA profiles identify involvement of Creb1 and Sec14l3 in bronchial epithelial changes in allergic asthma

View Article: PubMed Central - PubMed

ABSTRACT

Asthma is highly prevalent, but current therapies cannot influence the chronic course of the disease. It is thus important to understand underlying early molecular events. In this study, we aimed to use microRNAs (miRNAs) - which are critical regulators of signaling cascades - to identify so far uncharacterized asthma pathogenesis pathways. Therefore, deregulation of miRNAs was assessed in whole lungs from mice with ovalbumin (OVA)-induced allergic airway inflammation (AAI). In silico predicted target genes were confirmed in reporter assays and in house-dust-mite (HDM) induced AAI and primary human bronchial epithelial cells (NHBE) cultured at the air-liquid interface. We identified and validated the transcription factor cAMP-responsive element binding protein (Creb1) and its transcriptional co-activators (Crtc1-3) as targets of miR-17, miR-144, and miR-21. Sec14-like 3 (Sec14l3) - a putative target of Creb1 - was down-regulated in both asthma models and in NHBE cells upon IL13 treatment, while it’s expression correlated with ciliated cell development and decreased along with increasing goblet cell metaplasia. Finally, we propose that Creb1/Crtc1-3 and Sec14l3 could be important for early responses of the bronchial epithelium to Th2-stimuli. This study shows that miRNA profiles can be used to identify novel targets that would be overlooked in mRNA based strategies.

No MeSH data available.


Pulmonary miRNA profile and selection of Creb1 as target gene.(a) Fold changes of miRNA expression analyzed by Exiqon microarray, TaqMan® microarray and qRT-PCR of lung homogenate of mice with OVA-induced allergic airway inflammation and healthy controls on d72 of the treatment protocol (n = 5 mice per group) with adjusted p-value. n.d.: not detected; - miRNA not available on the array; *minor strand of miRNA. (b) List of genes and miRNA-binding sites in their 3′-UTRs as predicted by five different algorithms (miRanda, PicTar, PITA, TargetSpy & TargetScanS). Darker color depicts more binding sites for the same miRNA.
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f1: Pulmonary miRNA profile and selection of Creb1 as target gene.(a) Fold changes of miRNA expression analyzed by Exiqon microarray, TaqMan® microarray and qRT-PCR of lung homogenate of mice with OVA-induced allergic airway inflammation and healthy controls on d72 of the treatment protocol (n = 5 mice per group) with adjusted p-value. n.d.: not detected; - miRNA not available on the array; *minor strand of miRNA. (b) List of genes and miRNA-binding sites in their 3′-UTRs as predicted by five different algorithms (miRanda, PicTar, PITA, TargetSpy & TargetScanS). Darker color depicts more binding sites for the same miRNA.

Mentions: Female 6–8 week old Balb/c mice were intraperitoneally (i.p.) sensitized five times with ovalbumin (OVA) (d7, d14, d28, d42 and d56) (Sigma Aldrich, St. Louis, USA) or phosphate buffered saline (PBS) and challenged two times (d70 and d71) with 1% OVA aerosol to induce allergic airway inflammation. Changes in pulmonary miRNA expression were analyzed by comparing OVA/OVA exposed mice with control mice (PBS/OVA) via locked nucleic acid (LNATM) microarray (Exiqon, Vedbaek, Germany). Of the 580 analyzed miRNAs, 55% were expressed in lung tissue and 37 showed a statistically significant (cut-off p-value < 10−4) altered expression in OVA-treated animals (Fig. 1a). The high number of “primary hits” was validated and confirmed by a PCR-based, quantitative low density array (LDA) measuring 518 murine miRNAs (TaqMan, Life Technologies, Carlsbad, USA) (Fig. 1a). From the 37 significantly regulated miRNAs (LNATM microarray) six miRNAs were not present on the LDA array, thus could not be measured, and three miRNAs were below the detection limit of the LDA array. Five miRNAs (miR-290, miR-720, miR-29c, miR-152 and miR-101a) showed inverse expressions, whereas 23 candidates showed comparable results (r = 0.62, p ≤ 0.0005; Spearman rho). Quantitative real time PCRs (qRT-PCR) of the six most significantly (p ≤ 3.9 × 10–9 – 1.47 × 10−14) dysregulated miRNAs with fold-changes >1.5 further corroborated the results of both arrays (Fig. 1a).


Pulmonary microRNA profiles identify involvement of Creb1 and Sec14l3 in bronchial epithelial changes in allergic asthma
Pulmonary miRNA profile and selection of Creb1 as target gene.(a) Fold changes of miRNA expression analyzed by Exiqon microarray, TaqMan® microarray and qRT-PCR of lung homogenate of mice with OVA-induced allergic airway inflammation and healthy controls on d72 of the treatment protocol (n = 5 mice per group) with adjusted p-value. n.d.: not detected; - miRNA not available on the array; *minor strand of miRNA. (b) List of genes and miRNA-binding sites in their 3′-UTRs as predicted by five different algorithms (miRanda, PicTar, PITA, TargetSpy & TargetScanS). Darker color depicts more binding sites for the same miRNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5382551&req=5

f1: Pulmonary miRNA profile and selection of Creb1 as target gene.(a) Fold changes of miRNA expression analyzed by Exiqon microarray, TaqMan® microarray and qRT-PCR of lung homogenate of mice with OVA-induced allergic airway inflammation and healthy controls on d72 of the treatment protocol (n = 5 mice per group) with adjusted p-value. n.d.: not detected; - miRNA not available on the array; *minor strand of miRNA. (b) List of genes and miRNA-binding sites in their 3′-UTRs as predicted by five different algorithms (miRanda, PicTar, PITA, TargetSpy & TargetScanS). Darker color depicts more binding sites for the same miRNA.
Mentions: Female 6–8 week old Balb/c mice were intraperitoneally (i.p.) sensitized five times with ovalbumin (OVA) (d7, d14, d28, d42 and d56) (Sigma Aldrich, St. Louis, USA) or phosphate buffered saline (PBS) and challenged two times (d70 and d71) with 1% OVA aerosol to induce allergic airway inflammation. Changes in pulmonary miRNA expression were analyzed by comparing OVA/OVA exposed mice with control mice (PBS/OVA) via locked nucleic acid (LNATM) microarray (Exiqon, Vedbaek, Germany). Of the 580 analyzed miRNAs, 55% were expressed in lung tissue and 37 showed a statistically significant (cut-off p-value < 10−4) altered expression in OVA-treated animals (Fig. 1a). The high number of “primary hits” was validated and confirmed by a PCR-based, quantitative low density array (LDA) measuring 518 murine miRNAs (TaqMan, Life Technologies, Carlsbad, USA) (Fig. 1a). From the 37 significantly regulated miRNAs (LNATM microarray) six miRNAs were not present on the LDA array, thus could not be measured, and three miRNAs were below the detection limit of the LDA array. Five miRNAs (miR-290, miR-720, miR-29c, miR-152 and miR-101a) showed inverse expressions, whereas 23 candidates showed comparable results (r = 0.62, p ≤ 0.0005; Spearman rho). Quantitative real time PCRs (qRT-PCR) of the six most significantly (p ≤ 3.9 × 10–9 – 1.47 × 10−14) dysregulated miRNAs with fold-changes >1.5 further corroborated the results of both arrays (Fig. 1a).

View Article: PubMed Central - PubMed

ABSTRACT

Asthma is highly prevalent, but current therapies cannot influence the chronic course of the disease. It is thus important to understand underlying early molecular events. In this study, we aimed to use microRNAs (miRNAs) - which are critical regulators of signaling cascades - to identify so far uncharacterized asthma pathogenesis pathways. Therefore, deregulation of miRNAs was assessed in whole lungs from mice with ovalbumin (OVA)-induced allergic airway inflammation (AAI). In silico predicted target genes were confirmed in reporter assays and in house-dust-mite (HDM) induced AAI and primary human bronchial epithelial cells (NHBE) cultured at the air-liquid interface. We identified and validated the transcription factor cAMP-responsive element binding protein (Creb1) and its transcriptional co-activators (Crtc1-3) as targets of miR-17, miR-144, and miR-21. Sec14-like 3 (Sec14l3) - a putative target of Creb1 - was down-regulated in both asthma models and in NHBE cells upon IL13 treatment, while it&rsquo;s expression correlated with ciliated cell development and decreased along with increasing goblet cell metaplasia. Finally, we propose that Creb1/Crtc1-3 and Sec14l3 could be important for early responses of the bronchial epithelium to Th2-stimuli. This study shows that miRNA profiles can be used to identify novel targets that would be overlooked in mRNA based strategies.

No MeSH data available.