Untersuchung von miRNAs mithilfe getrockneter Blutstropfen

Due to their connection to a great diversity of diseases and their prevalence in blood, microRNAs (miRNAs) are envisaged as biomarkers in liquid biopsy diagnostics. Utilizing dried blood spots (DBS) for the isolation of miRNAs greatly facilitates both the sample collection and the storage in comparison to liquid blood. MiRNAs isolated from DBS can be used for the analysis of individual miRNAs and for high-throughput analyses of the entire miRNome

Emerging concepts of miRNA therapeutics: from cells to clinic

MicroRNAs (miRNAs) are very powerful genetic regulators, as evidenced by the fact that a single miRNA can direct entire cellular pathways via interacting with a broad spectrum of target genes. This property renders miRNAs as highly interesting therapeutic tools to restore cell functions that are altered as part of a disease phenotype. However, this strength of miRNAs is also a weakness because their cellular effects are so numerous that off-target effects can hardly be avoided. In this review, we point out the main challenges and the strategies to specifically address the problems that need to be surmounted in the push toward a therapeutic application of miRNAs. Particular emphasis is given to approaches that have already found their way into clinical studies

Dynamik und Wirkung der microRNA-Expression in der frühen Phase der humanen CD4+ T-Zell-Aktivierung

T-Zellen spielen eine unverzichtbare Rolle im Rahmen der adaptiven Immunabwehr (JANEWAY et al., 1975; MATTER, 1974; ZHU, 2018). Eine veränderte T-Zell-Aktivität wird im Zusammenhang mit der Entstehung verschiedener schwerwiegender Erkrankungen diskutiert (CHEN, JOHN WHERRY, 2020; DORNMAIR et al., 2003; MOLON et al., 2016; TAN et al., 2020). Für die Regulation der T-Zell-Aktivierung scheint die Expression von microRNAs von zentraler Bedeutung zu sein (EICHMULLER et al., 2017; KOPP et al., 2013; LIU et al., 2013b; LORENZI et al., 2012). Als Biomarker für T-Zell gekoppelte Erkrankungen und als Instrument für die gezielten Manipulation der T-Zell-Funktion könnten microRNAs in Zukunft im diagnostischen und therapeutischen Bereich Anwendungen finden (COLAMATTEO et al., 2019; LONG et al., 2018). Umfangreiche Kenntnisse zur Expression und zur spezifischen Wirkungsweise von microRNAs sind dafür eine wesentliche Voraussetzung (EICHMULLER et al., 2017; GIRI et al., 2019; JI et al., 2016; RUPAIMOOLE, SLACK, 2017). Die Ziele dieser Arbeit bestanden in der Erfassung, Quantifizierung und funktionellen Charakterisierung von microRNA-Expressionsveränderungen in der frühen 24 h Phase der humanen T-Zell-Aktivierung. Die Studie wurde an CD4+ T-Zellen durchgeführt, die den größten Anteil der T-Zell-Subtypen im menschlichen Blut bilden und von zentraler Bedeutung für die Effektivität der Immunantwort sind (LUCKHEERAM et al., 2012; TOLLERUD et al., 1989). Als Ausgangspunkt der Analysen wurden detaillierte zeitaufgelöste Hochdurchsatz-Datensätze zur microRNA- und mRNA-Expression über die initialen 24 h der in vitro induzierten CD4+ T-Zell-Aktivierung erstellt. Auf Basis der umfassenden Zeitverlaufsdaten konnten 39 zentrale microRNAs mit deutlicher Expressionsveränderung im Kontext des T-Zell-Aktivierungsprozesses identifiziert werden. Hierbei konnte eine hohe technische und interindividuelle Reproduzierbarkeit der microRNA-Zeitverlaufsveränderungen gezeigt werden. Die Klassifizierung der zeitaufgelösten Expressionsprofile ermöglichte Rückschlüsse zu den Mechanismen, die zur Koordination der microRNA-Expression im Rahmen des frühen T-Zell-Aktivierungsprozesses beitragen könnten. Dazu zählt beispielsweise die Transkriptionskontrolle multipler microRNAs durch den Transkriptionsfaktor c-Myc. Durch die Etablierung einer neuen Methodik zur Quantifizierung der microRNA-Expression konnte ein grundlegender Überblick über die aktivierungs-gekoppelten Expressionsänderungen für alle 815 detektierten microRNAs gewonnen werden. Für die zentral veränderte miR-155-5p wurde ein molekularer microRNA-Expressionsbereich zwischen 7,54×104 und 3,25×106 Molekülen pro Nanogramm zellulärer Gesamt-RNA ermittelt. Im Hinblick auf eine zukünftige therapeutische microRNA-Nutzung konnte somit ein Rahmen für eine physiologisch angepasste Dosierung gelegt werden (LAI et al., 2019; RUPAIMOOLE, SLACK, 2017). Um Einblicke in die Wirkungsweise von microRNAs zu gewinnen, wurden die regulatorischen Funktionen der miR-155-5p untersucht, indem in silico vorhergesagte Zielgene mit invers korrelierenden mRNA-Zeitverlaufsdaten anhand von dualen Luciferase-Reporter-Assays getestet wurden. Dabei konnte die microRNA-Zielgen-Interaktion für 17 von 19 getesteten Genen bestätigt und der regulatorische Einfluss der microRNA für ein breites Spektrum zellulärer Funktionen gezeigt werden. Als Hinweis auf mögliche kooperative microRNA-Funktionen im Kontext des T-Zell-Aktivierungsprozesses konnten durch die in silico Analyse von microRNA-Zielgen-Netzwerken drei zentral veränderte microRNA-Paare mit multiplen gemeinsamen Zielgenen identifiziert werden (let-7b-5p/miR-26a-5p, miR-17-5p/miR-20a-5p und miR-21-5p/miR-155-5p). Exemplarisch für das miR-21-5p/miR-155-5p-Paar konnte im Rahmen von dualen Luciferase-Assays ein synergistischer regulatorischer Effekt auf das LEMD3-Zielgen nachgewiesen werden. Die Untersuchungen dieser Arbeit geben Einblicke in die Dynamik und Wirkung der microRNA-Expression im Kontext des frühen CD4+ T-Zell-Aktivierungsprozesses und liefern eine umfassende Datengrundlage zur Aufklärung von microRNA-regulierten Signalnetzwerken sowie für die Entwicklung von microRNA-basierten Therapieansätzen.T cells play an indispensable role for the adaptive immune defense (JANEWAY et al., 1975; MATTER, 1974; ZHU, 2018). An altered T cell activity is associated with the emergence of severe diseases (CHEN, JOHN WHERRY, 2020; DORNMAIR et al., 2003; MOLON et al., 2016; TAN et al., 2020). The expression of microRNAs appears to be critically important for the regulation of T cell activation (EICHMULLER et al., 2017; KOPP et al., 2013; LIU et al., 2013b; LORENZI et al., 2012). MicroRNAs bear high potential as future biomarkers for T cell related pathologies and as a tool for the targeted manipulation of T cells (COLAMATTEO et al., 2019; LONG et al., 2018). A fundamental understanding of the expression and functionality of microRNAs is a principal prerequisite to apply microRNAs in a future diagnostic and therapeutic context (EICHMULLER et al., 2017; GIRI et al., 2019; JI et al., 2016; RUPAIMOOLE, SLACK, 2017). The objectives of this thesis were the detection, quantification and functional characterization of microRNA expression changes within the early 24 h phase of human T cell activation. The study was conducted on CD4+ T cells, which constitute the largest fraction of T cell subtypes in human blood and are central to the effectiveness of the immune response (LUCKHEERAM et al., 2012; TOLLERUD et al., 1989). First, detailed time-resolved high-throughput datasets on microRNA and mRNA expression were determined during the initial 24 h of in vitro induced CD4+ T cell activation. Based on the comprehensive time-course data in context of the T cell activation process, 39 central microRNAs with distinct expression changes were identified and a very high technical and inter-individual reproducibility of the microRNA time-course changes was demonstrated. The classification of the time-resolved expression profiles allowed conclusions about the mechanisms that likely coordinate the microRNA expression during the early T cell activation process, as for example the transcriptional control of multiple microRNAs by the transcription factor c-Myc. A newly implemented methodology to quantify the microRNA expression allowed to determine both the molecular range of microRNA expression and the activation-coupled expression changes for all of the 815 detected microRNAs. For the most prominently changed miR-155-5p a microRNA expression range between 7.54×104 and 3.25×106 molecules per nanogram of cellular total RNA was determined. These quantitative data constitute a basis to define a physiological dosage for a future therapeutic application of microRNAs (LAI et al., 2019; RUPAIMOOLE, SLACK, 2017). To gain further insights into the functionality of microRNAs, regulatory functions of miR-155-5p were characterized by the testing of target genes that were in silico predicted and showed inverse correlating mRNA time-course data. Dual luciferase reporter assays confirmed microRNA target interactions for 17 out of the 19 tested genes. The regulatory microRNA impact was demonstrated on a broad range of cellular functions. In silico analyses of microRNA-target networks identified three prominently changed microRNA pairs with multiple shared targets (let-7b-5p/miR-26a-5p, miR-17-5p/miR-20a-5p and miR-21-5p/miR-155-5p), indicating presumable cooperative microRNA functions in the context of the T cell activation process. Exemplary for the miR-21-5p/miR-155-5p pair, dual luciferase assays demonstrated a synergistic regulatory effect on the LEMD3 target gene. This thesis provides insights into the dynamics and functionality of microRNA expression during the early CD4+ T cell activation process and a comprehensive data basis for both the elucidation of microRNA regulated signaling networks and the development of microRNA-based therapeutic approaches

miR-34a-5p as molecular hub of pathomechanisms in Huntington's disease

Background Although a pivotal role of microRNA (miRNA, miR) in the pathogenesis of Huntington’s disease (HD) is increasingly recognized, the molecular functions of miRNAs in the pathomechanisms of HD await further elucidation. One of the miRNAs that have been associated with HD is miR-34a-5p, which was deregulated in the mouse R6/2 model and in human HD brain tissues. Methods The aim of our study was to demonstrate interactions between miR-34a-5p and HD associated genes. By computational means we predicted 12 801 potential target genes of miR-34a-5p. An in-silico pathway analysis revealed 22 potential miR-34a-5p target genes in the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway “Huntington’s disease”. Results Using our high-throughput miRNA interaction reporter assay (HiTmIR) we identifed NDUFA9, TAF4B, NRF1, POLR2J2, DNALI1, HIP1, TGM2 and POLR2G as direct miR-34a-5p target genes. Direct binding of miR-34a-5p to target sites in the 3’UTRs of TAF4B, NDUFA9, HIP1 and NRF1 was verifed by a mutagenesis HiTmIR assay and by determining endogenous protein levels for HIP1 and NDUFA9. STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) analysis identifed protein–protein interaction networks associated with HD like “Glutamine Receptor Signaling Pathway” and “Calcium Ion Transmembrane Import Into Cytosol”. Conclusion Our study demonstrates multiple interactions between miR-34a-5p and HD associated target genes and thereby lays the ground for future therapeutic interventions using this miRNA

Induction of the Endoplasmic-Reticulum-Stress Response: MicroRNA-34a Targeting of the IRE1α-Branch

Neurodegenerative disorders such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) are characterized by the accumulation of misfolded proteins in the endoplasmic reticulum (ER) and the unfolded protein response (UPR). Modulating the UPR is one of the major challenges to counteract the development of neurodegenerative disorders and other diseases with affected UPR. Here, we show that miR-34a-5p directly targets the IRE1α branch of the UPR, including the genes BIP, IRE1α, and XBP1. Upon induction of ER stress in neuronal cells, miR-34a-5p overexpression impacts the resulting UPR via a significant reduction in IRE1α and XBP1s that in turn leads to decreased viability, increased cytotoxicity and caspase activity. The possibility to modify the UPR signaling pathway by a single miRNA that targets central genes of the IRE1α branch offers new perspectives for future therapeutic approaches against neurodegeneration

Wrinkle in the plan: miR-34a-5p impacts chemokine signaling by modulating CXCL10/CXCL11/CXCR3-axis in CD4+, CD8+ T cells, and M1 macrophages

Background In 2016 the first-in-human phase I study of a miRNA-based cancer therapy with a liposomal mimic of microRNA-34a-5p (miR-34a-5p) was closed due to five immune related serious adverse events (SAEs) resulting in four patient deaths. For future applications of miRNA mimics in cancer therapy it is mandatory to unravel the miRNA effects both on the tumor tissue and on immune cells. Here, we set out to analyze the impact of miR-34a-5p over-expression on the CXCL10/CXCL11/CXCR3 axis, which is central for the development of an effective cancer control. Methods We performed a whole genome expression analysis of miR-34a-5p transfected M1 macrophages followed by an over-representation and a protein–protein network analysis. In-silico miRNA target prediction and dual luciferase assays were used for target identification and verification. Target genes involved in chemokine signaling were functionally analyzed in M1 macrophages, CD4+ and CD8+ T cells. Results A whole genome expression analysis of M1 macrophages with induced miR-34a-5p over-expression revealed an interaction network of downregulated target mRNAs including CXCL10 and CXCL11. In-silico target prediction in combination with dual luciferase assays identified direct binding of miR-34a-5p to the 3′UTRs of CXCL10 and CXCL11. Decreased CXCL10 and CXCL11 secretion was shown on the endogenous protein level and in the supernatant of miR-34a-5p transfected and activated M1 macrophages. To complete the analysis of the CXCL10/CXCL11/CXCR3 axis, we activated miR-34a-5p transfected CD4+ and CD8+ T cells by PMA/Ionomycin and found reduced levels of endogenous CXCR3 and CXCR3 on the cell surface. Conclusions MiR-34a-5p mimic administered by intravenous administration will likely not only be up-taken by the tumor cells but also by the immune cells. Our results indicate that miR-34a-5p over-expression leads in M1 macrophages to a reduced secretion of CXCL10 and CXCL11 chemokines and in CD4+ and CD8+ T cells to a reduced expression of CXCR3. As a result, less immune cells will be attracted to the tumor site. Furthermore, high levels of miR-34a-5p in naive CD4+ T cells can in turn hinder Th1 cell polarization through the downregulation of CXCR3 leading to a less pronounced activation of cytotoxic T lymphocytes, natural killer, and natural killer T cells and possibly contributing to lymphocytopenia

Masitinib as an adjunct therapy for mild-to-moderate Alzheimer's disease: a randomised, placebo-controlled phase 2 trial

International audienceIntroductionNeuroinflammation is thought to be important in Alzheimer's disease pathogenesis. Mast cells are a key component of the inflammatory network and participate in the regulation of the blood-brain barrier's permeability. Masitinib, a selective oral tyrosine kinase inhibitor, effectively inhibits the survival, migration and activity of mast cells. As the brain is rich in mast cells, the therapeutic potential of masitinib as an adjunct therapy to standard care was investigated.MethodsA randomised, placebo-controlled, phase 2 study was performed in patients with mild-to-moderate Alzheimer's disease, receiving masitinib as an adjunct to cholinesterase inhibitor and/or memantine. Patients were randomly assigned to receive masitinib (n = 26) (starting dose of 3 or 6 mg/kg/day) or placebo (n = 8), administered twice daily for 24 weeks. The primary endpoint was change from baseline in the Alzheimer's Disease Assessment Scale - cognitive subscale (ADAS-Cog) to assess cognitive function and the related patient response rate.ResultsThe rate of clinically relevant cognitive decline according to the ADAS-Cog response (increase >4 points) after 12 and 24 weeks was significantly lower with masitinib adjunctive treatment compared with placebo (6% vs. 50% for both time points; P = 0.040 and P = 0.046, respectively). Moreover, whilst the placebo treatment arm showed worsening mean ADAS-Cog, Alzheimer's Disease Cooperative Study Activities of Daily Living Inventory, and Mini-Mental State Examination scores, the masitinib treatment arm reported improvements, with statistical significance between treatment arms at week 12 and/or week 24 (respectively, P = 0.016 and 0.030; P = 0.035 and 0.128; and P = 0.047 and 0.031). The mean treatment effect according to change in ADAS-Cog score relative to baseline at weeks 12 and 24 was 6.8 and 7.6, respectively. Adverse events occurred more frequently with masitinib treatment (65% vs. 38% of patients); however, the majority of events were of mild or moderate intensity and transitory. Severe adverse events occurred at a similar frequency in the masitinib and placebo arms (15% vs. 13% of patients, respectively). Masitinib-associated events included gastrointestinal disorders, oedema, and rash.ConclusionsMasitinib administered as add-on therapy to standard care during 24 weeks was associated with slower cognitive decline in Alzheimer's disease, with an acceptable tolerance profile. Masitinib may therefore represent an innovative avenue of treatment in Alzheimer's disease. This trial provides evidence that may support a larger placebo-controlled investigation.Trial registrationClinicaltrials.gov NCT0097611

Modulation of intracellular calcium signaling by microRNA-34a-5p

Adjusting intracellular calcium signaling is an important feature in the regulation of immune cell function and survival. Here we show that miR-34a-5p, a small non-coding RNA that is deregulated in many common diseases, is a regulator of store-operated Ca2+ entry (SOCE) and calcineurin signaling. Upon miR-34a-5p overexpression, we observed both a decreased depletion of ER calcium content and a decreased Ca2+ influx through Ca2+ release-activated Ca2+ channels. Based on an in silico target prediction we identified multiple miR-34a-5p target genes within both pathways that are implicated in the balance between T-cell activation and apoptosis including ITPR2, CAMLG, STIM1, ORAI3, RCAN1, PPP3R1, and NFATC4. Functional analysis revealed a decrease in Ca2+ activated calcineurin pathway activity measured by a reduced IL-2 secretion due to miR-34a-5p overexpression. Impacting SOCE and/or downstream calcineurin/NFAT signaling by miR-34a-5p offers a possible future approach to manipulate immune cells for clinical interventions

Quantitative and time-resolved miRNA pattern of early human T cell activation

T cells are central to the immune response against various pathogens and cancer cells. Complex networks of transcriptional and post-transcriptional regulators, including microRNAs (miRNAs), coordinate the T cell activation process. Available miRNA datasets, however, do not sufficiently dissolve the dynamic changes of miRNA controlled networks upon T cell activation. Here, we established a quantitative and time-resolved expression pattern for the entire miRNome over a period of 24 h upon human Tcell activation. Based on our time-resolved datasets, we identified central miRNAs and specified common miRNA expression profiles. We found the most prominent quantitative expression changes for miR155-5p with a range from initially 40 molecules/cell to 1600 molecules/cell upon T-cell activation. We established a comprehensive dynamic regulatory network of both the up- and downstream regulation of miR155. Upstream, we highlight IRF4 and its complexes with SPI1 and BATF as central for the transcriptional regulation of miR-155. Downstream of miR-155-5p, we verified 17 of its target genes by the time-resolved data recorded after T cell activation. Our data provide comprehensive insights into the range of stimulus induced miRNA abundance changes and lay the ground to identify efficient points of intervention for modifying the T cell response

Low miR-150-5p and miR-320b Expression Predicts Reduced Survival of COPD Patients

Chronic obstructive pulmonary disease (COPD) is associated with an increased risk of death, reducing life expectancy on average between 5 and 7 years. The survival time after diagnosis, however, varies considerably as a result of the heterogeneity of COPD. Therefore, markers that predict individual survival of COPD patients are of great value. We analyzed baseline molecular profiles and collected 54 months of follow-up data of the cohort study “COPD and SYstemic consequences-COmorbidities NETwork” (COSYCONET). Genome-wide microRNA signatures from whole blood collected at time of the inclusion in the study were generated for 533 COPD patients including patients that deceased during the 54-month follow-up period (n = 53) and patients that survived this period (n = 480). We identified two blood-born microRNAs (miR-150-5p and miR-320b) that were highly predictive for survival of COPD patients. The expression change was then confirmed by RT-qPCR in 245 individuals. Ninety percent of patients with highest expression of miR-150-5p survived the 54-month period in contrast to only 50% of patients with lowest expression intensity. Moreover, the abundance of the oncogenic miR-150-5p in blood of COPD patients was predictive for the development of cancer. Thus, molecular profiles measured at the time of a COPD diagnosis have a high predictive power for the survival of patients