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Cystic fibrosis (CF), a genetic disorder, is characterized by chronic lung disease. Small non-coding RNAs are key regulators of gene expression and participate in various processes, which are dysregulated in CF; however, they remain poorly studied. Here, we determined the complete microRNAs (miRNAs) expression pattern in three CF ex vivo models. The miRNA profiles of air-liquid interface cultures of airway epithelia (bronchi, nasal cells, and nasal polyps) samples from patients with CF and non-CF controls were obtained by deep sequencing. Compared with non-CF controls, several miRNAs were deregulated in CF samples; for instance, miR-181a-5p and the miR-449 family were upregulated. Moreover, mature miRNAs often showed variations (i.e. isomiRs) relative to their reference sequence, such as miR-101, suggesting that miRNAs consist of heterogeneous repertoires of multiple isoforms with different effects on gene expression. Analysis of miR-181a-5p and miR-101-3p roles indicated that they regulate the expression of WISP1, a key component of cell proliferation/migration programs. We showed that miR-101 and miR-181a-5p participated in aberrant recapitulation of wound healing programs by controlling WISP1 mRNA and protein level. Our miRNA expression data bring new insights into CF physiopathology and define new potential therapeutic targets in CF. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

International audience; Impaired airway homeostasis in chronic obstructive pulmonary disease (COPD) could be partly related to club cell secretory protein (CCSP) deficiency. We hypothesize that CCSP G38A polymorphism is involved and aim to examine the influence of the CCSP G38A polymorphism on CCSP transcription levels and its regulatory mechanisms. CCSP genotype and CCSP levels in serum and sputum were assessed in 66 subjects with stable COPD included in a 1-yr observational study. Forty-nine of them had an exacerbation. In an in vitro study, the impact on the CCSP promoter of 38G wild-type or 38A variant was assessed. BEAS-2B cells were transfected by either the 38G or 38A construct, in the presence/absence of cigarette smoke extract (CSE) or lipopolysaccharides (LPS). Cotransfections with modulating transcription factors, p53 and Nkx2.1, identified by in silico analysis by using ConSite and TFSEARCH were conducted. A allele carrier COPD patients had lower serum and sputum CCSP levels, especially among active smokers, and a decreased body mass index, airflow obstruction, dyspnea, and exercise capacity (BODE) score. In vitro, baseline CCSP transcription levels were similar between the wild and variant constructs. CSE decreased more profoundly the CCSP transcription level of 38A transfected cells. The opposite effect was observed with p53 cotransfection. LPS stimulation induced CCSP repression in 38A promoter transfected cells. Cotransfection with Nkx2.1 significantly activated the CCSP promoters irrespective of the polymorphism. Circulating CCSP levels are associated with smoking and the CCSP G38A polymorphism. CSE, LPS, and the Nkx2.1 and p53 transcription factors modulated the CCSP promoter efficiency. The 38A polymorphism exaggerated the CCSP repression in response to p53 and CSE.

International audience; PURPOSE:Although 97-99% of CFTR mutations have been identified, great efforts must be made to detect yet-unidentified mutations.METHODS:We developed a small-scale next-generation sequencing approach for reliably and quickly scanning the entire gene, including noncoding regions, to identify new mutations. We applied this approach to 18 samples from patients suffering from cystic fibrosis (CF) in whom only one mutation had hitherto been identified.RESULTS:Using an in-house bioinformatics pipeline, we could rapidly identify a second disease-causing CFTR mutation for 16 of 18 samples. Of them, c.1680-883A>G was found in three unrelated CF patients. Analysis of minigenes and patients' transcripts showed that this mutation results in aberrantly spliced transcripts because of the inclusion of a pseudoexon. It is located only three base pairs from the c.1680-886A>G mutation (1811+1.6kbA>G), the fourth most frequent mutation in southwestern Europe. We next tested the effect of antisense oligonucleotides targeting splice sites on these two mutations on pseudoexon skipping. Oligonucleotide transfection resulted in the restoration of the full-length, in-frame CFTR transcript, demonstrating the effect of antisense oligonucleotide-induced pseudoexon skipping in CF.CONCLUSION:Our data confirm the importance of analyzing noncoding regions to find unidentified mutations, which is essential to designing targeted therapeutic approaches.

Club cell secretory protein (CCSP) is an endogenous anti-inflammatory product with a protective role in the respiratory tract. Impaired airway homeostasis in chronic bronchial diseases might be related to CCSP G38A polymorphism but the SNP-transcription relationship is not established. This study was to examine the influence of ccsp G38A polymorphism on CCSP expression and regulation. Vectors constructions (proximal promoter: 229bp; and distal promoter: 926bp) containing -38G wild type or the -38A variant were designed. Human bronchial BEAS-2B cells were transfected with these proximal or distal promoters, in presence/absence of cigarette smoke extract (CSE). Dual luciferase assay system reflected CCSP transcription levels. In parallel, dual transfections with candidate modulating transcription factors identified by in silico analysis using ConSite and TFSEARCH were conducted. No significant changes are induced by the -38G and -38A vectors in transiently transfected BEAS-2B cells. Cotransfection with Nkx2.1 significantly increased CCSP transcription. Interestingly, with proximal promoter only,-38A variant gene response to Nkx2.1 was higher than -38G wild type response. CSE decreased CCSP transcription. In depth-in silico analyzes identified two novel candidate transcription factors that could specifically bind to the G38A locus: Thing1E47 and p53. Our data indicate that two novel transcription factors and CSE could affect CCSP expression. Also, -38A variant CCSP gene modulation induced by Nkx2.1 is significant. Finding transcription factors that could restore normal levels of CCSP expression is of interest.

International audience; Cystic Fibrosis (CF) is a common autosomal recessive disorder, caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. CFTR gene expression is tightly controlled by transcriptional and post-transcriptional regulatory factors, resulting in complex spatial and temporal expression patterns. Here, we describe an overview of the findings about the contribution of ncRNAs, especially miRNAs, in physiological CFTR gene expression and in CF. Determination of mechanisms governing its expression is essential for developing new CF therapies. ncRNAs, including lncRNAs and miRNAs, could also contribute to CF progression and severity and their dysregulation in CF opens new perspectives for patient follow-up and treatment.

TheCFTRgene displays a tightly regulated tissue-specific and temporal expression. Mutations in this gene cause cystic fibrosis (CF). In this study we wanted to identifytrans-regulatory elements responsible forCFTRdifferential expression in fetal and adult lung, and to determine the importance of inhibitory motifs in theCFTR-3′UTR with the aim of developing new tools for the correction of disease-causing mutations withinCFTR.We show that lung development-specific transcription factors (FOXA, C/EBP) and microRNAs (miR-101, miR-145, miR-384) regulate the switch from strong fetal to very lowCFTRexpression after birth. By using miRNome profiling and gene reporter assays, we found that miR-101 and miR-145 are specifically upregulated in adult lung and that miR-101 directly acts on its cognate site in theCFTR-3′UTR in combination with an overlapping AU-rich element. We then designed miRNA-binding blocker oligonucleotides (MBBOs) to prevent binding of several miRNAs to theCFTR-3′UTR and tested them in primary human nasal epithelial cells from healthy individuals and CF patients carrying the p.Phe508delCFTRmutation. These MBBOs rescued CFTR channel activity by increasing CFTR mRNA and protein levels.Our data offer new understanding of the control of theCFTRgene regulation and new putative correctors for cystic fibrosis.

Considering that the extensive study of the CFTR gene classically performed in molecular diagnosis does not detect all disease-causing mutations, we previously developed an approach for a complete resequencing of the CFTR locus to search for mutations deeply located in introns (Bonini et al. Genet Med, 2015). After identifying candidate intronic mutations, we now aim to restore full-length CFTR transcripts by using antisense oligonucleotides also named Target Site Blockers (TSB). DNA from 15 patients with only one CF mutation were collected through a national collaborative study. Enrichments of the CFTR locus (250 kb) by long-range PCR or targeted capture were sequenced using 454 GS Junior and MiSeq Illumina platforms. TSB (Exiqon) were specifically designed for the deleterious variants. Out of 200 variations detected, our in-house pipeline pointed out 10 intronic variations, per DNA in average. In silico analysis tools predicted that 13 variants (for the 15 patients) had a deleterious effect on CFTR splicing by promoting inclusion of pseudoexons. TSB were tested whether the splicing defect was confirmed. For instance, TSB used on bronchial and primary nasal cells transfected with minigene constructs, significantly restored the full-length CFTR transcript for the well-known mutation c.1680–886A>G and a new identified c.1680–883A>G. Two new mutations in introns 18 and 23 are being tested. Finally, our massively parallel sequencing strategy lead to the identification of new CF-causing mutations in introns. Our findings also demonstrate the efficiency of antisense oligonucleotides for a targeted therapeutic for cystic fibrosis. Work supported by Vaincre La Mucoviscidose.

Objectives: CFTR gene, described as a housekeeping gene, displays a tightly temporal and developmental regulation. In lung, CFTR transcripts are abundant during fetal compared to adult stage, where only two copies per cell are detected. The aim of this work is the characterization of regulatory elements acting on the amount of CFTR transcripts (transcription and stability) to explain the regulation of CFTR gene expression in lung. Methods: 3′UTR and 5′UTR regions of CFTR gene were analysed thanks to bioinformatics tools (TS Search, Consite, AREsite, miRBase). To evaluate the importance of each predicted motif for transcription factors (FTs) and microRNAs (miRNAs), functional studies were realized (Luciferase assays, siRNA, RTq-PCR, constructions containing or not degenerated motifs for trans-regulators. . . ). Based on the identification of miRNAs binding sites on CFTR mRNA, we also propose to test new potential therapeutic tools for CF patients by designing oligonucleotides TSB (Target Site Blockers). Results: We characterized new motifs for the binding of FTs on CFTR mRNA, key regulators in the pulmonary development (FOX, C/EBP, Nkx2), and miRNAs (miR101, miR145) explaining the modulation of CFTR transcrits rate in lung. In addition, the first findings on the use of oligonucleotides TSB led to increased of CFTR mRNA and protein level in CF patients. Conclusion:We propose a molecular network involving FTs and miRNAs to explain difference in the CFTR transcrit level, in fetal and adult lungs. Identification of cisregulatory motifs led to envision new tools for CF therapy. Supported by Vaincre la mucoviscidose.