Target Site Blockers Research Articles

EPS1.1 CFTR-specific target site blocker nanoparticles as a potential CF gene therapy strategy

EPS1.1 CFTR-specific target site blocker nanoparticles as a potential CF gene therapy strategy

Inhibition of miR-181a protects female mice from transient focal cerebral ischemia by targeting astrocyte estrogen receptor-α.

Whether the effect of miR-181a is sexually dimorphic in stroke is unknown. Prior work showed protection of male mice with miR-181a inhibition. Estrogen receptor-α (ERα) is an identified target of miR181 in endometrium. Therefore we investigated the separate and joint effects of miR-181a inhibition and 17β-estradiol (E2) replacement after ovariectomy. Adult female mice were ovariectomized and implanted with an E2- or vehicle-containing capsule for 14d prior to 1h middle cerebral artery occlusion (MCAO). Each group received either miR-181a antagomir or mismatch control by intracerebroventricular injection 24h before MCAO. After MCAO neurologic deficit and infarct volume were assessed. Primary male and female astrocyte cultures were subjected to glucose deprivation with miR-181a inhibitor or transfection control, and E2 or vehicle control, with/without ESRα knockdown with small interfering RNA. Cell death was assessed by propidium iodide staining, and lactate dehydrogenase assay. A miR-181a/ERα target site blocker (TSB), with/without miR-181a mimic, was used to confirm targeting of ERα by miR-181a in astrocytes. Individually, miR-181a inhibition or E2 decreased infarct volume and improved neurologic score in female mice, and protected male and female astrocyte cultures. Combined miR-181a inhibition plus E2 afforded greater protection of female mice and female astrocyte cultures, but not in male astrocyte cultures. MiR-181a inhibition only increased ERα levels in vivo and in female cultures, while ERα knockdown with siRNA increased cell death in both sexes. Treatment with ERα TSB was strongly protective in both sexes. In conclusion, the results of the present study suggest miR-181a inhibition enhances E2-mediated stroke protection in females in part by augmenting ERα production, a mechanism detected in female mice and female astrocytes. Sex differences were observed with combined miR-181a inhibition/E2 treatment, and miR-181a targeting of ERα.

MiR-155-5p positively regulates CCL17-induced colon cancer cell migration by targeting RhoA.

Colorectal cancer is the second most common cause of cancer-related death, which is due to migration of tumor cells to distant sites of metastasis. Accumulating data indicate that mciroRNAs play an important role in several aspects of colon cancer cell biology. Herein, we examined the role of miR-155-5p in colon cancer cell migration induced by the CCL17-CCR4 axis in HT-29 colon cancer cells. We found that miR-155-5p knockdown in serum starved colon cancer cells decreased CCL17-induced cell chemotaxis. Moreover, knocking down miR-155-5p markedly decreased CCL17-provoked activation of RhoA in colon cancer cells. Bioinformatics analysis predicted two putative binding sites in the AU-rich element at the 3′-UTR of RhoA mRNA. MiR-155-5p binding to RhoA mRNA was verified using a target site blocker and functionally validated by RNA immunoprecipitation assays, showing that miR-155-5p-dependent regulation of RhoA mRNA is mediated by AU-rich elements present in the 3′-UTR region. Taken together, these results show that miR-155-5p positively regulates RhoA mRNA levels and translation as well as cell migration in serum starved colon cancer cells and indicate that targeting miR-155-5p might be a useful strategy to antagonize colon cancer metastasis.

MicroRNA-101 Regulates Multiple Developmental Programs to Constrain Excitation in Adult Neural Networks

A critical feature of neural networks is that they balance excitation and inhibition to prevent pathological dysfunction. How this is achieved is largely unknown, although deficits in the balance contribute to many neurological disorders. We show here that a microRNA (miR-101) is a key orchestrator of this essential feature, shaping the developing network to constrain excitation in the adult. Transient early blockade of miR-101 induces long-lasting hyper-excitability and persistent memory deficits. Using target site blockers invivo, we identify multiple developmental programs regulated in parallel by miR-101 to achieve balanced networks. Repression of one target, NKCC1, initiates the switch in γ-aminobutyric acid (GABA) signaling, limits early spontaneous activity, and constrains dendritic growth. Kif1a and Ank2 are targeted to prevent excessive synapse formation. Simultaneous de-repression of these three targets completely phenocopies major dysfunctions produced by miR-101 blockade. Our results provide new mechanistic insight into brain development and suggest novel candidates for therapeutic intervention.

Corrigendum: Endothelial Dicer promotes atherosclerosis and vascular inflammation by miRNA-103-mediated suppression of KLF4.

Nature Communications 7: Article number: 10521 (2016); Published: 3 February 2016; Updated: 7 June 2016 This Article contains an error in the dose of LNA-target site blocker oligonucleotides that was administered. In the Methods section, under the subheading ‘In vivo target site blocker treatment’ the dose ‘0.

WS17.2 Identification of CF mutations in deep intronic regions: Design of antisense oligonucleotides for a targeted therapeutic approach

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.

Abstract 17767: A MiR-31-dependent Loss of Dystrophin & Nnos in the Human Atria Plays a Key Role in Atrial Fibrillation-induced Electrical Remodelling

Background: Management of atrial fibrillation (AF) remains a challenge. AF remodels the atrial electrical properties, thereby increasing resistance to treatment. Although remodeling has long been a target for therapeutic intervention in AF, the mechanisms driving this phenomenon are incompletely understood. Methods & Results: Using atrial samples from 351 patients (73 AF), and 67 goats (34 AF), we show that atrial-specific upregulation of miR31 causes dystrophin (DYS) translational repression and accelerates mRNA degradation of neuronal nitric oxide synthase (nNOS) leading to a profound reduction in NO availability. Six prediction algorithms and reporter assays established DYS and nNOS as miR-31 targets. In atrial myocytes from patients with AF (hAFm), both DYS and nNOS bind to miR-31 within the RNA induced silencing complex (RISC). In actinomycin D-treated myocytes from patients in sinus rhythm (hSRm), miR31 accelerated nNOS (but not DYS) mRNA decay. mRNA of nNOS (but not DYS) & protein content of nNOS, DYS & DYS associated proteins were significantly reduced in hAFm. Inhibition of miR31 in hAFm restored both DYS & nNOS protein. Protection of the nNOS miR31 binding site with a target site blocker (TSB) restored nNOS mRNA and protein (but not DYS), whereas, DYS-TSB increased both DYS and nNOS protein (but not mRNA), in keeping with an effect of DYS restoration on nNOS protein stability. Indeed, K48-linked polyubiquitination of nNOS was increased in hAFm & prevented by proteasome inhibition with MG132. NOS: inhibition (SMTC, 100 nmol) or transfection with a miR31 mimic was employed to evaluate the impact of these findings on atrial electrical properties. Both interventions shortened action potential duration (APD90) and abolished rate-dependency of the APD90 in hSRm but not in hAFm. By contrast, miR31 inhibition restored APD & APD rate-dependency in hAFm (both reversed by SMTC) but had no effect on hSRm. In mice (n=126), nNOS gene deletion or inhibition shortened atrial APD90 & lead to a 2-fold increase in AF inducibility in response to atrial burst pacing. Conclusions: These findings identify atrial-specific upregulation of miR31 in human AF as a key mechanism causing atrial loss of dystrophin and nNOS, which, in turn, lead to the electrical phenotype begetting AF.

The tumour suppressor miR-34c targets MET in prostate cancer cells

Background:The microRNA, miR-34c, is a well-established regulator of tumour suppression. It is downregulated in most forms of cancers and inhibits malignant growth by repressing genes involved in processes such as proliferation, anti-apoptosis, stemness, and migration. We have previously reported downregulation and tumour suppressive properties for miR-34c in prostate cancer (PCa).Methods:In this study, we set out to further characterize the mechanisms by which miR-34c deregulation contributes to PCa progression. The genes regulated by miR-34c in the PCa cell line PC3 were identified by microarray analyses and were found to be enriched in cell death, cell cycle, cellular growth, and cellular movement pathways. One of the identified targets was MET, a receptor tyrosine kinase activated by hepatocyte growth factor, that is crucial for metastatic progression.Results:We confirmed the inhibitory effect of miR-34c on both MET transcript and protein levels. The binding of miR-34c to two binding sites in the 3′-UTR of MET was validated using luciferase reporter assays and target site blockers. The effect of this regulation on the miR-34c inhibition of the migratory phenotype was also confirmed. In addition, a significant inverse correlation between miR-34c expression levels and MET immunostaining was found in PCa patients.Conclusion:These findings provide a novel molecular mechanism of MET regulation in PCa and contribute to the increasing evidence that miR-34c has a key tumour suppressive role in PCa.

WS20.1 Role of transcription factors and microRNAs in CFTR gene expression

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.