Limits...
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.


IL13 treatment of NHBE cells reduces CREB1 and SEC14l3.(a) PAS staining of transwell membranes. (b) qRT-PCR for MUC5AC. (c) qRT-PCR for CREB1 and CRTC1-3. (d) Representative western blot and densitometrical analysis. The blot was cropped to improve clarity – full-length blots are provided in Supplemental Fig. S5 (e) qRT-PCR for SEC14L3 and FOXJ1. All graphs depict fold change vs. control. n.d. = not detectable. All n = 4, Mann-Whitney U, *p < 0.05 vs. untreated control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5382551&req=5

f7: IL13 treatment of NHBE cells reduces CREB1 and SEC14l3.(a) PAS staining of transwell membranes. (b) qRT-PCR for MUC5AC. (c) qRT-PCR for CREB1 and CRTC1-3. (d) Representative western blot and densitometrical analysis. The blot was cropped to improve clarity – full-length blots are provided in Supplemental Fig. S5 (e) qRT-PCR for SEC14L3 and FOXJ1. All graphs depict fold change vs. control. n.d. = not detectable. All n = 4, Mann-Whitney U, *p < 0.05 vs. untreated control.

Mentions: Induction of goblet cell metaplasia was confirmed by a cross-section PAS staining of the transwell membranes (Fig. 7a) and Mucin 5A/C (MUC5AC) mRNA increase (Fig. 7b). The mRNA of CREB1 and its co-activator CRTCs decreased significantly after only 24 hours of IL13 treatment, but then increased over time (Fig. 7c). These findings were largely translated to the protein level (Fig. 7d), although with more prolonged decreases. SEC14L3 and FOXJ1 transcript levels were significantly decreased upon IL13 treatment (with levels not detectable at 24 h), abolishing the increase observed during normal differentiation (Fig. 7e).


Pulmonary microRNA profiles identify involvement of Creb1 and Sec14l3 in bronchial epithelial changes in allergic asthma
IL13 treatment of NHBE cells reduces CREB1 and SEC14l3.(a) PAS staining of transwell membranes. (b) qRT-PCR for MUC5AC. (c) qRT-PCR for CREB1 and CRTC1-3. (d) Representative western blot and densitometrical analysis. The blot was cropped to improve clarity – full-length blots are provided in Supplemental Fig. S5 (e) qRT-PCR for SEC14L3 and FOXJ1. All graphs depict fold change vs. control. n.d. = not detectable. All n = 4, Mann-Whitney U, *p < 0.05 vs. untreated control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: IL13 treatment of NHBE cells reduces CREB1 and SEC14l3.(a) PAS staining of transwell membranes. (b) qRT-PCR for MUC5AC. (c) qRT-PCR for CREB1 and CRTC1-3. (d) Representative western blot and densitometrical analysis. The blot was cropped to improve clarity – full-length blots are provided in Supplemental Fig. S5 (e) qRT-PCR for SEC14L3 and FOXJ1. All graphs depict fold change vs. control. n.d. = not detectable. All n = 4, Mann-Whitney U, *p < 0.05 vs. untreated control.
Mentions: Induction of goblet cell metaplasia was confirmed by a cross-section PAS staining of the transwell membranes (Fig. 7a) and Mucin 5A/C (MUC5AC) mRNA increase (Fig. 7b). The mRNA of CREB1 and its co-activator CRTCs decreased significantly after only 24 hours of IL13 treatment, but then increased over time (Fig. 7c). These findings were largely translated to the protein level (Fig. 7d), although with more prolonged decreases. SEC14L3 and FOXJ1 transcript levels were significantly decreased upon IL13 treatment (with levels not detectable at 24 h), abolishing the increase observed during normal differentiation (Fig. 7e).

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.