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A Peptide Nucleic Acid against MicroRNA miR-145-5p Enhances the Expression of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in Calu-3 Cells.

ABSTRACT: Peptide nucleic acids (PNAs) are very useful tools for gene regulation at different levels, but in particular in the last years their use for targeting microRNA (anti-miR PNAs) has provided impressive advancements. In this respect, microRNAs related to the repression of cystic fibrosis transmembrane conductance regulator (CFTR) gene, which is defective in cystic fibrosis, are of great importance in the development of new type of treatments. In this paper we propose the use of an anti-miR PNA for targeting miR-145, a microRNA reported to suppress CFTR expression. Octaarginine-anti-miR PNA conjugates were delivered to Calu-3 cells, exerting sequence dependent targeting of miR-145-5p. This allowed to enhance expression of the miR-145 regulated CFTR gene, analyzed at mRNA (RT-qPCR, Reverse Transcription quantitative Polymerase Chain Reaction) and CFTR protein (Western blotting) level.

SUBMITTER: Fabbri E

PROVIDER: S-EPMC6017273 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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A Peptide Nucleic Acid (PNA) Masking the miR-145-5p Binding Site of the 3'UTR of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) mRNA Enhances CFTR Expression in Calu-3 Cells.

Project description:Peptide nucleic acids (PNAs) have been demonstrated to be very useful tools for gene regulation at different levels and with different mechanisms of action. In the last few years the use of PNAs for targeting microRNAs (anti-miRNA PNAs) has provided impressive advancements. In particular, targeting of microRNAs involved in the repression of the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which is defective in cystic fibrosis (CF), is a key step in the development of new types of treatment protocols. In addition to the anti-miRNA therapeutic strategy, inhibition of miRNA functions can be reached by masking the miRNA binding sites present within the 3'UTR region of the target mRNAs. The objective of this study was to design a PNA masking the binding site of the microRNA miR-145-5p present within the 3'UTR of the CFTR mRNA and to determine its activity in inhibiting miR-145-5p function, with particular focus on the expression of both CFTR mRNA and CFTR protein in Calu-3 cells. The results obtained support the concept that the PNA masking the miR-145-5p binding site of the CFTR mRNA is able to interfere with miR-145-5p biological functions, leading to both an increase of CFTR mRNA and CFTR protein content.

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High-Potency Phenylquinoxalinone Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Activators.

Project description:We previously identified phenylquinoxalinone CFTRact-J027 (4) as a cystic fibrosis transmembrane conductance regulator (CFTR) activator with an EC50 of ?200 nM and demonstrated its therapeutic efficacy in mouse models of constipation. Here, structure-activity studies were done on 36 synthesized phenylquinoxalinone analogs to identify compounds with improved potency and altered metabolic stability. Synthesis of the phenylquinoxalinone core was generally accomplished by condensation of 1,2-phenylenediamines with substituted phenyloxoacetates. Structure-activity studies established, among other features, the privileged nature of a properly positioned nitro moiety on the 3-aryl group. Synthesized analogs showed improved CFTR activation potency compared to 4 with EC50 down to 21 nM and with greater metabolic stability. CFTR activators have potential therapeutic indications in constipation, dry eye, cholestatic liver diseases, and inflammatory lung disorders.

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Project description:Most cases of cystic fibrosis (CF) are caused by mutations that block the biosynthetic maturation of the CF gene product, the CF transmembrane conductance regulator (CFTR) chloride channel. CFTR-processing mutants fail to escape the endoplasmic reticulum and are rapidly degraded. Current efforts to induce the maturation of CFTR mutants target components of the biosynthetic pathway (e.g., chaperones) rather than CFTR per se. Such methods are inherently nonspecific. Here we show that the most common CF-causing mutant (DeltaF508-CFTR) can form mature, functional chloride channels that reach the cell surface when coexpressed with several other CFTR-processing mutants or with amino fragments of the wild-type CFTR protein. This transcomplementation effect required a specific match between the region flanking the disease-causing mutation and the complementing fragment; e.g., amino fragments complemented DeltaF508-CFTR but not H1085R (a carboxy-processing mutant), whereas a carboxy fragment complemented H1085R but not DeltaF508-CFTR. Transcomplementing fragments did not affect CFTR interactions with Hsc70, a chaperone previously implicated in CFTR biosynthesis. Instead, they may promote CFTR maturation by blocking nonproductive interactions between domains within the same or neighboring CFTR polypeptides that prevent normal processing. These findings indicate that it may be possible to develop CF therapies (e.g., mini-cDNA constructs for gene therapy) that are tailored to specific disease-causing mutants of CFTR.

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Transcriptional adaptation to cystic fibrosis transmembrane conductance regulator deficiency

Project description:Cystic fibrosis, the most commonly inherited lethal pulmonary disorder in Caucasians, is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR). To identify genomic responses to the presence or absence of CFTR in pulmonary tissues in vivo, microarray analyses of lung mRNAs were performed on whole lung tissue from mice lacking (CFTR(-)) or expressing mouse CFTR (CFTR(+)). Whereas the histology of lungs from CFTR(-) and CFTR(+) mice was indistinguishable, statistically significant increases in the relative abundance of 29 and decreases in 25 RNAs were identified by RNA microarray analysis. Of RNAs whose expression was consistently altered by the absence of CFTR, functional classes of genes influencing gene transcription, inflammation, intracellular trafficking, signal transduction, and ion transport were identified. RNAs encoding the transcription factor CCAAT enhancer-binding protein (CEBP) delta and interleukin (IL) 1beta, both known to regulate CFTR expression, were induced, perhaps indicating adaptation to the lack of CFTR. RNAs mediating lung inflammation including calgranulin-S100 family members, IL-1beta and IL-4, were increased. Likewise, expression of several membrane transport proteins that interact directly with CFTR were increased, suggesting that CFTR-protein complexes initiate genomic responses. Absence of CFTR influenced the expression of genes modulating diverse pulmonary cell functions that may ameliorate or contribute to the pathogenesis of CF. Keywords: Genotype comparison

2006-03-24 | GSE4513 | GEO

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| S-EPMC3029685 | biostudies-literature

A Review of Trikafta: Triple Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Modulator Therapy.

Project description:Cystic fibrosis (CF) is a potentially fatal genetic disease that causes serious lung damage. With time, researchers have a more complete understanding of the molecular-biological defects that underlie CF. This knowledge is leading to alternative approaches regarding the treatment of this condition. Trikafta is the third FDA-approved drug that targets the F508del mutation of the CFTR gene. The drug is a combination of three individual drugs which are elexacaftor (ELX), tezacaftor (TEZ), and ivacaftor (IVA). This trio increases the activity of the cystic fibrosis transmembrane conductance regulator (CFTR) protein and reduces the mortality and morbidity rates in CF patients. The effectiveness of Trikafta, seen in clinical trials, outperforms currently available therapies in terms of lung function, quality of life, sweat chloride reduction, and pulmonary exacerbation reduction. The safety and efficacy of CFTR modulators in children with CF have also been studied. Continued evaluation of patient data is needed to confirm its long-term safety and efficacy. In this study, we will focus on reviewing data from clinical trials regarding the benefits of CFTR modulator therapy. We address the impact of Trikafta on lung function, pulmonary exacerbations, and quality of life. Adverse events of the different CFTR modulators are discussed.

| S-EPMC8266292 | biostudies-literature

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