11 research outputs found
A peptide nucleic acid against microrna miR-145-5p enhances the expression of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in Calu-3 Cells
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
Cellular Uptakes, Biostabilities and Anti-miR-210 Activities of Chiral Arginine-PNAs in Leukaemic K562 Cells
A series of 18-mer peptide nucleic acids (PNAs) targeted
against micro-RNA miR-210 was synthesised and tested in a cellular system. Unmodified PNAs, R8-conjugated PNAs and modified PNAs c ontaining eight arginine residues on the backbone, either as C2-modified (R) or C5-modified (S) monomers, all with the same sequence, were compared. Two different models were used for the modified PNAs: one with alternated chiral and achiral monomers and one with a stretch of chiral monomers at the N terminus. The melting temperatures of these
derivatives were found to be extremely high and 5m urea was
used to assess differences between the different structures.
FACS analysis and qRT-PCR on K562 chronic myelogenous leukaemic
cells indicated that arginine-conjugated and backbonemodified
PNAs display good cellular uptake, with best performances
for the C2-modified series. Resistance to enzymatic
degradation was found to be higher for the backbone-modified
PNAs, thus enhancing the advantage of using these derivatives
rather than conjugated PNAs in the cells in serum, and
this effect is magnified in the presence of peptidases such as
trypsin. Inhibition of miR-210 activity led to changes in the
erythroid differentiation pathway, which were more evident in
mithramycin-treated cells. Interestingly, the anti-miR activities
differed with use of different PNAs, thus suggesting a role of
the substituents not only in the cellular uptake, but also in the
mechanism of miR recognition and inactivation. This is the first
report relating to the use of backbone-modified PNAs as antimiR
agents. The results clearly indicate that backbone-modified
PNAs are good candidates for the development of very efficient
drugs based on anti-miR activity, due to their enhanced
bioavailabilities, and that overall anti-miR performance is a
combination of cellular uptake and RNA binding
Targeting microRNAs involved in human diseases: A novel approach for modification of gene expression and drug development
The identification of all epigenetic modifications (i.e. DNA methylation, histone modifications and
expression of noncoding RNAs such as microRNAs) involved in gene regulation is one of the major steps
forward for understanding human biology in both normal and pathological conditions and for
development of novel drugs. In this context, microRNAs play a pivotal role. This review article focuses on
the involvement of microRNAs in the regulation of gene expression, on the possible role of microRNAs in
the onset and development of human pathologies, and on the pharmacological alteration of the
biological activity of microRNAs. RNA and DNA analogs, which can selectively target microRNAs using
Watson–Crick base pairing schemes, provide a rational and efficient way to modulate gene expression.
These compounds, termed antago-miR or anti-miR have been described in many examples in the recent
literature and have proved to be able to perform regulatory as well as therapeutic functions. Among
these, a still not fully exploited class is that of peptide nucleic acids (PNAs), promising tools for the
inhibition of miRNA activity, with important applications in gene therapy and in drug development.
PNAs targeting miR-122, miR-155 and miR-210 have already been developed and their biological effects
studied both in vitro and in vivo
Treatment of human airway epithelial Calu-3\ua0cells with a peptide-nucleic acid (PNA) targeting the microRNA miR-101-3p is associated with increased expression of the cystic fibrosis Transmembrane Conductance Regulator () gene
Since the identification of microRNAs (miRNAs) involved in the regulation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, miRNAs known to down-regulate the expression of the CFTR and associated proteins have been investigated as potential therapeutic targets. Here we show that miR-101-3p, targeting the 3'-UTR sequence of the CFTR mRNA, can be selectively inhibited by a peptide nucleic acid (PNA) carrying a full complementary sequence. With respect to clinical relevance of microRNA targeting, it is expected that reduction in concentration of miRNAs (the anti-miRNA approach) could be associated with increasing amounts of target mRNAs. Consistently to this hypothesis, we report that PNA-mediated inhibition of miR-101-3p was accompanied by CFTR up-regulation. Next Generation Sequencing (NGS) was performed in order to verify the effects of the anti-miR-101-3p PNA on the Calu-3 miRNome. Upon inhibition of miR-101-3p we observed a fold change (FC) expression <2 of the majority of miRNAs (403/479, 84.13%), whereas we identified a list of dysregulated miRNAs, suggesting that specific miRNA inhibition (in our case miR-101-3p) might be accompanied by alteration of expression of other miRNAs, some of them known to be involved in Cystic Fibrosis (CF), such as miR-155-5p and miR-125b-5p
A peptide-nucleic acid targeting miR-335-5p enhances expression of cystic fibrosis transmembrane conductance regulator (CFTR) gene with the possible involvement of the CFTR scaffolding protein NHERF1
(1) Background: Up-regulation of the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR) might be of great relevance for the development of therapeutic protocols for cystic fibrosis (CF). MicroRNAs are deeply involved in the regulation of CFTR and scaffolding proteins (such as NHERF1, NHERF2 and Ezrin). (2) Methods: Content of miRNAs and mRNAs was analyzed by RT-qPCR, while the CFTR and NHERF1 production was analyzed by Western blotting. (3) Results: The results here described show that the CFTR scaffolding protein NHERF1 can be up-regulated in bronchial epithelial Calu-3 cells by a peptide-nucleic acid (PNA) targeting miR-335-5p, predicted to bind to the 3'-UTR sequence of the NHERF1 mRNA. Treatment of Calu-3 cells with this PNA (R8-PNA-a335) causes also up-regulation of CFTR. (4) Conclusions: We propose miR-335-5p targeting as a strategy to increase CFTR. While the efficiency of PNA-based targeting of miR-335-5p should be verified as a therapeutic strategy in CF caused by stop-codon mutation of the CFTR gene, this approach might give appreciable results in CF cells carrying other mutations impairing the processing or stability of CFTR protein, supporting its application in personalized therapy for precision medicine
High levels of apoptosis are induced in human glioma cell lines by co-administration of peptide nucleic acids targeting miR-221 and miR-222
The biological activity of a combined treatment of U251, U373 and T98G glioma cell lines with two anti-miR PNAs, directed against miR‑221 and miR‑222 and conjugated with an ocataarginine tail (R8-PNA-a221 and R8-PNA-a222) for efficient cellular delivery, was determined. Apoptosis was analyzed, and the effect of the combined treatment of glioma cells with either or both PNAs on the reversion of drug-resistance phenotype was assessed in the temozolomide-resistant T98G glioma cell line. Selectivity of PNA/miRNA interactions was studied by surface plasmon resonance (SPR)-based Biacore analysis. Specificity of the PNA effects at the cellular level was analyzed by RT-qPCR. These experiments support the concept that the effects of R8-PNA-a221 and R8-PNA-a222 are specific. The studies on apoptosis confirmed that the R8-PNA-a221 induces apoptosis and demonstrated the pro-apoptotic effects of R8-PNA-a222. Remarkably, increased pro-apoptotic effects were obtained with the co-administration of both anti-miR‑221 and anti-miR‑222 PNAs. In addition, co-administration of R8-PNA-a221 and R8-PNA-a222 induced apoptosis of TMZ-treated T98G cells at a level higher than that obtained following singular administration of R8-PNA-a221 or R8-PNA-a222
Health services utilization in patients with eating disorders: evidence from a cohort study in Emilia-Romagna
Purpose: To estimate the treated prevalence of eating disorders (ED) in Emilia-Romagna, Italy, and to compare health services utilization among age groups and geographical areas. Methods: The study cohort consists of patients aged 12\u201364 years with a primary or secondary ED diagnosis, treated in regional healthcare facilities in 2012. Patients were followed up for 1\ua0year from the first contact. Data were extracted from regional administrative databases. Results: The study cohort included 1550 cases, 36.8\ua0% with anorexia nervosa, 21.9\ua0% with bulimia nervosa and 41.3\ua0% with ED not otherwise specified. Adolescents\ua0(12\u201317 years) were 18.6\ua0%, young adults\ua0(18\u201330) 32.7\ua0% and older adults\ua0(31\u201364) 48.7\ua0%. The annual treated prevalence rate was 5.2/10,000 (13.3 for adolescents, 9.3 for young adults and 3.4 for older adults) and was highest among adolescent (24.6/10,000) and young adult females (17.1/10,000). Cases without a record for ED in the previous year were 46.8\ua0%. Older adults displayed higher comorbidity and used more services including hospital-based care. Outpatient care greatly exceeded inpatient care across age groups. Variations in care patterns across regional areas were found. Conclusions: Our results indicate that the care pathway for ED varies among age groups and geographical areas, but is consistent with the regional care model that favors the use of outpatient services. Future perspectives include evaluating the integration among mental health services, specialty outpatient units and primary care