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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 increases during NHBE differentiation and correlates with FoxJ1.(a) Treatment scheme of differentiation of primary normal human bronchial epithelial cells (NHBE) into a pseudostratified epithelium at the air-liquid interface. (b) qRT-PCR for SEC14L3 and FOXJ1 from day 7 to day28 of differentiation (n = 4 independent experiments). y-axis depicts dCp values (Cpgene-Cpreference gene) – higher dCp means lower expression (reversed scale). (c) Linear correlation of dCp values of SEC14L3 and FOXJ1 (n = 4 independent experiments).
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f6: SEC14L3 increases during NHBE differentiation and correlates with FoxJ1.(a) Treatment scheme of differentiation of primary normal human bronchial epithelial cells (NHBE) into a pseudostratified epithelium at the air-liquid interface. (b) qRT-PCR for SEC14L3 and FOXJ1 from day 7 to day28 of differentiation (n = 4 independent experiments). y-axis depicts dCp values (Cpgene-Cpreference gene) – higher dCp means lower expression (reversed scale). (c) Linear correlation of dCp values of SEC14L3 and FOXJ1 (n = 4 independent experiments).

Mentions: In order to get further insight into the regulation of epithelial SEC14L3 in the context of allergic airway inflammation, we differentiated primary normal human bronchial epithelial (NHBE) cells (Lonza, Basel, Switzerland) at the air-liquid interface in PneumaCult™-ALI medium (Stemcell Technologies; Köln, Germany) containing 1% penicillin/streptomycin at 5% CO2 and 37 °C. After 28 days, a pseudostratified epithelium was formed, containing goblet, club, ciliated and basal cells34. To induce early asthma-like changes in the bronchial epithelium, the cells were treated with 10 ng/mL of the potent Th2 cytokine IL13 (R&D Systems, Wiesbaden, Germany) from the basolateral side between d0 and d7 (Fig. 6a).


Pulmonary microRNA profiles identify involvement of Creb1 and Sec14l3 in bronchial epithelial changes in allergic asthma
SEC14L3 increases during NHBE differentiation and correlates with FoxJ1.(a) Treatment scheme of differentiation of primary normal human bronchial epithelial cells (NHBE) into a pseudostratified epithelium at the air-liquid interface. (b) qRT-PCR for SEC14L3 and FOXJ1 from day 7 to day28 of differentiation (n = 4 independent experiments). y-axis depicts dCp values (Cpgene-Cpreference gene) – higher dCp means lower expression (reversed scale). (c) Linear correlation of dCp values of SEC14L3 and FOXJ1 (n = 4 independent experiments).
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Related In: Results  -  Collection

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f6: SEC14L3 increases during NHBE differentiation and correlates with FoxJ1.(a) Treatment scheme of differentiation of primary normal human bronchial epithelial cells (NHBE) into a pseudostratified epithelium at the air-liquid interface. (b) qRT-PCR for SEC14L3 and FOXJ1 from day 7 to day28 of differentiation (n = 4 independent experiments). y-axis depicts dCp values (Cpgene-Cpreference gene) – higher dCp means lower expression (reversed scale). (c) Linear correlation of dCp values of SEC14L3 and FOXJ1 (n = 4 independent experiments).
Mentions: In order to get further insight into the regulation of epithelial SEC14L3 in the context of allergic airway inflammation, we differentiated primary normal human bronchial epithelial (NHBE) cells (Lonza, Basel, Switzerland) at the air-liquid interface in PneumaCult™-ALI medium (Stemcell Technologies; Köln, Germany) containing 1% penicillin/streptomycin at 5% CO2 and 37 °C. After 28 days, a pseudostratified epithelium was formed, containing goblet, club, ciliated and basal cells34. To induce early asthma-like changes in the bronchial epithelium, the cells were treated with 10 ng/mL of the potent Th2 cytokine IL13 (R&D Systems, Wiesbaden, Germany) from the basolateral side between d0 and d7 (Fig. 6a).

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.