Small RNA and transcriptome sequencing reveal the role of miR-199a-3p in inflammatory processes in cystic fibrosis airways.

Cystic fibrosis (CF) is the most common lethal genetic disease, caused by CFTR (cystic fibrosis transmembrane conductance regulator) gene mutations. CF is characterized by an ionic imbalance and thickened mucus, which impair mucociliary clearance and promote bacterial colonization and the establishment of infection/inflammation cycles. However, the origin of this inflammation remains unclear, although microRNAs (miRNAs) are suspected to be involved. MiRNAs are small non-coding RNAs that bind to the 3'-untranslated regions (UTRs) of target gene mRNA, thereby repressing their translation and/or inducing their degradation. The goal of this study was to investigate the role of microRNAs associated with pulmonary inflammation in CF patients. Through the analysis of all miRNAs (miRNome) in human primary air-liquid interface cultures, we demonstrated that miR-199a-3p is the only miRNA downregulated in CF patients compared to controls. Moreover, through RNA sequencing (transcriptome) analysis, we showed that 50% of all deregulated mRNAs are linked directly or indirectly to the NF-κB pathway. To identify a specific target, we used bioinformatics analysis to predict whether miR-199a-3p targets the 3'-UTR of IKBKB, which encodes IKKß, a major protein in the NF-κB pathway. Subsequently, we used bronchial explants from CF patients to show that miR-199a-3p expression is downregulated compared to controls and inversely correlated with increases in expression of IKKß and IL-8. Through functional studies, we showed that miR-199a-3p modulates the expression of IKBKB through a direct interaction at its 3'-UTR in bronchial epithelial cells from CF patients. In miR-199a-3p overexpression experiments, we demonstrated that for CF cells, miR-199a-3p reduced IKKß protein expression, NF-κB activity, and IL-8 secretion. Taken together, our findings show that miR-199a-3p plays a negative regulatory role in the NF-κB signalling pathway and that its low expression in CF patients contributes to chronic pulmonary inflammation. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Lidocaine Administration Controls MicroRNAs Alterations Observed After Lung Ischemia-Reperfusion Injury.

BACKGROUND: Ischemia-reperfusion injury (IRI) is associated with morbidity and mortality. MicroRNAs (miRNAs) have emerged as regulators of IRI, and they are involved in the pathogenesis of organ rejection. Lidocaine has proven anti-inflammatory activity in several tissues but its modulation of miRNAs has not been investigated. This work aims to investigate the involvement of miRNAs in lung IRI in a lung auto-transplantation model and to investigate the effect of lidocaine. METHODS: Three groups (sham, control, and Lidocaine), each comprising 6 pigs, underwent a lung autotransplantation. All groups received the same anesthesia. In addition, animals of lidocaine group received a continuous intravenous administration of lidocaine (1.5 mg/kg/h) during surgery. Lung biopsies were taken before pulmonary artery clamp, before reperfusion, 30 minutes postreperfusion (Rp-30), and 60 minutes postreperfusion (Rp-60). Samples were analyzed for different miRNAs (miR-122, miR-145, miR-146a, miR-182, miR-107, miR-192, miR-16, miR-21, miR-126, miR-127, miR142-5p, miR152, miR155, miR-223, and let7) via the use of reverse-transcription quantitative polymerase chain reaction. Results were normalized with miR-103. RESULTS: The expression of miR-127 and miR-16 did not increase after IRI. Let-7d, miR-21, miR-107, miR-126, miR-145, miR-146a, miR-182, and miR-192 significantly increased at the Rp-60 (control versus sham P < .001). miR-142-5p, miR-152, miR-155, and miR 223 significantly increased at the Rp-30 (control versus sham P < .001) and at the Rp-60 (control versus. sham P < .001). The administration of lidocaine was able to attenuate these alterations in a significant way (control versus Lidocaine P < .001). CONCLUSIONS: Lung IRI caused dysregulation miRNA. The administration of lidocaine reduced significantly miRNAs alterations.

Identification of Paired-related Homeobox Protein 1 as a key mesenchymal transcription factor in pulmonary fibrosis.

Matrix remodeling is a salient feature of idiopathic pulmonary fibrosis (IPF). Targeting cells driving matrix remodeling could be a promising avenue for IPF treatment. Analysis of transcriptomic database identified the mesenchymal transcription factor PRRX1 as upregulated in IPF. PRRX1, strongly expressed by lung fibroblasts, was regulated by a TGF-ß/PGE2 balance in vitro in control and IPF human lung fibroblasts, while IPF fibroblast-derived matrix increased PRRX1 expression in a PDGFR-dependent manner in control ones. PRRX1 inhibition decreased human lung fibroblast proliferation by downregulating the expression of S phase cyclins. PRRX1 inhibition also impacted TGF-ß driven myofibroblastic differentiation by inhibiting SMAD2/3 phosphorylation through phosphatase PPM1A upregulation and TGFBR2 downregulation, leading to TGF-ß response global decrease. Finally, targeted inhibition of Prrx1 attenuated fibrotic remodeling in vivo with intra-tracheal antisense oligonucleotides in bleomycin mouse model of lung fibrosis and ex vivo using human and mouse precision-cut lung slices. Our results identified PRRX1 as a key mesenchymal transcription factor during lung fibrogenesis.

Chaotic activation of developmental signalling pathways drives idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterised by an important remodelling of lung parenchyma. Current evidence indicates that the disease is triggered by alveolar epithelium activation following chronic lung injury, resulting in alveolar epithelial type 2 cell hyperplasia and bronchiolisation of alveoli. Signals are then delivered to fibroblasts that undergo differentiation into myofibroblasts. These changes in lung architecture require the activation of developmental pathways that are important regulators of cell transformation, growth and migration. Among others, aberrant expression of profibrotic Wnt-ß-catenin, transforming growth factor-ß and Sonic hedgehog pathways in IPF fibroblasts has been assessed. In the present review, we will discuss the transcriptional integration of these different pathways during IPF as compared with lung early ontogeny. We will challenge the hypothesis that aberrant transcriptional integration of these pathways might be under the control of a chaotic dynamic, meaning that a small change in baseline conditions could be sufficient to trigger fibrosis rather than repair in a chronically injured lung. Finally, we will discuss some potential opportunities for treatment, either by suppressing deleterious mechanisms or by enhancing the expression of pathways involved in lung repair. Whether developmental mechanisms are involved in repair processes induced by stem cell therapy will also be discussed.