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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.


Sec14l3 in murine allergic airway inflammation.(a) qRT-PCR and (b) Western blot and densitometrical analysis of OVA-treated animals, mean ± SD, (n = 5 mice/ group). The blot was cropped to improve clarity – full-length blots are provided in Supplemental Fig. S3 (c) Representative lung sections: Sec14l3 (red) vs. DAPI (blue), (left) or PAS (right). Arrowheads: Sec14l3 + cells (left) and goblet cells (right) (4 mice/group). HDM-treated mice (n = 6 mice/group), mean ± SD (d) qRT-PCR. (a,d) y-axis depicts dCp values (Cpgene-Cpreference gene) – higher dCp means lower expression (reversed scale) (e) densitometrical analysis of Western blot. *p < 0.05; **p < 0.01 vs. PBS. (f) Correlations of Sec14l3 mRNA (dCp values) with total cell counts in BAL and airway hyperreactivity (Ahr) as depicted by resistance to 12.5 mg/ml methacholine.
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f5: Sec14l3 in murine allergic airway inflammation.(a) qRT-PCR and (b) Western blot and densitometrical analysis of OVA-treated animals, mean ± SD, (n = 5 mice/ group). The blot was cropped to improve clarity – full-length blots are provided in Supplemental Fig. S3 (c) Representative lung sections: Sec14l3 (red) vs. DAPI (blue), (left) or PAS (right). Arrowheads: Sec14l3 + cells (left) and goblet cells (right) (4 mice/group). HDM-treated mice (n = 6 mice/group), mean ± SD (d) qRT-PCR. (a,d) y-axis depicts dCp values (Cpgene-Cpreference gene) – higher dCp means lower expression (reversed scale) (e) densitometrical analysis of Western blot. *p < 0.05; **p < 0.01 vs. PBS. (f) Correlations of Sec14l3 mRNA (dCp values) with total cell counts in BAL and airway hyperreactivity (Ahr) as depicted by resistance to 12.5 mg/ml methacholine.

Mentions: As Creb1 and the Crtcs were altered in the both murine AAI models, we asked whether their down-regulation also negatively influences the transcription of Creb1 target genes. To this end, we searched an mRNA array previously performed (using lung homogenate collected on d72) of our OVA model31 (GEO database, ID: GSE6496 (http://www.ncbi.nlm.nih.gov/geo) for down-regulated genes containing CRE-sites in OVA/OVA vs. PBS/OVA mice. Of 185 decreased genes, 35 contained putative CRE-elements (Table S2). We selected Sec14-like 3 (Sec14l3) for further investigation as it had already been found to be decreased in allergic airway inflammation in rats32 and to be specifically expressed in murine airway ciliated cells33. Sec14l3 mRNA levels were decreased in lungs of OVA-animals (Fig. 5a). Similar to Creb1 and the Crtcs, Sec14l3 protein levels increased in PBS/OVA mice, but the increase was abolished in animals with OVA-induced allergic inflammation (Fig. 5b). Immunofluorescence staining demonstrated Sec14l3 expression in airway ciliated cells of healthy animals, with lower levels in OVA-induced AAI (Fig. 5c). PAS staining of matching lung sections showed a concomitant goblet cell metaplasia (Fig. 5c). These findings were corroborated in HDM-induced AAI (Figs 5d,e and S4). In the HDM model, Sec14l3 mRNA levels also significantly and negatively correlated with increasing airway hyperreactivity and total cell counts in the BAL (Fig. 5f). This suggested that loss of Sec14l3 is associated with AAI, independent of the type of allergen.


Pulmonary microRNA profiles identify involvement of Creb1 and Sec14l3 in bronchial epithelial changes in allergic asthma
Sec14l3 in murine allergic airway inflammation.(a) qRT-PCR and (b) Western blot and densitometrical analysis of OVA-treated animals, mean ± SD, (n = 5 mice/ group). The blot was cropped to improve clarity – full-length blots are provided in Supplemental Fig. S3 (c) Representative lung sections: Sec14l3 (red) vs. DAPI (blue), (left) or PAS (right). Arrowheads: Sec14l3 + cells (left) and goblet cells (right) (4 mice/group). HDM-treated mice (n = 6 mice/group), mean ± SD (d) qRT-PCR. (a,d) y-axis depicts dCp values (Cpgene-Cpreference gene) – higher dCp means lower expression (reversed scale) (e) densitometrical analysis of Western blot. *p < 0.05; **p < 0.01 vs. PBS. (f) Correlations of Sec14l3 mRNA (dCp values) with total cell counts in BAL and airway hyperreactivity (Ahr) as depicted by resistance to 12.5 mg/ml methacholine.
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f5: Sec14l3 in murine allergic airway inflammation.(a) qRT-PCR and (b) Western blot and densitometrical analysis of OVA-treated animals, mean ± SD, (n = 5 mice/ group). The blot was cropped to improve clarity – full-length blots are provided in Supplemental Fig. S3 (c) Representative lung sections: Sec14l3 (red) vs. DAPI (blue), (left) or PAS (right). Arrowheads: Sec14l3 + cells (left) and goblet cells (right) (4 mice/group). HDM-treated mice (n = 6 mice/group), mean ± SD (d) qRT-PCR. (a,d) y-axis depicts dCp values (Cpgene-Cpreference gene) – higher dCp means lower expression (reversed scale) (e) densitometrical analysis of Western blot. *p < 0.05; **p < 0.01 vs. PBS. (f) Correlations of Sec14l3 mRNA (dCp values) with total cell counts in BAL and airway hyperreactivity (Ahr) as depicted by resistance to 12.5 mg/ml methacholine.
Mentions: As Creb1 and the Crtcs were altered in the both murine AAI models, we asked whether their down-regulation also negatively influences the transcription of Creb1 target genes. To this end, we searched an mRNA array previously performed (using lung homogenate collected on d72) of our OVA model31 (GEO database, ID: GSE6496 (http://www.ncbi.nlm.nih.gov/geo) for down-regulated genes containing CRE-sites in OVA/OVA vs. PBS/OVA mice. Of 185 decreased genes, 35 contained putative CRE-elements (Table S2). We selected Sec14-like 3 (Sec14l3) for further investigation as it had already been found to be decreased in allergic airway inflammation in rats32 and to be specifically expressed in murine airway ciliated cells33. Sec14l3 mRNA levels were decreased in lungs of OVA-animals (Fig. 5a). Similar to Creb1 and the Crtcs, Sec14l3 protein levels increased in PBS/OVA mice, but the increase was abolished in animals with OVA-induced allergic inflammation (Fig. 5b). Immunofluorescence staining demonstrated Sec14l3 expression in airway ciliated cells of healthy animals, with lower levels in OVA-induced AAI (Fig. 5c). PAS staining of matching lung sections showed a concomitant goblet cell metaplasia (Fig. 5c). These findings were corroborated in HDM-induced AAI (Figs 5d,e and S4). In the HDM model, Sec14l3 mRNA levels also significantly and negatively correlated with increasing airway hyperreactivity and total cell counts in the BAL (Fig. 5f). This suggested that loss of Sec14l3 is associated with AAI, independent of the type of allergen.

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.