84 research outputs found
Investigating the functional role of lncRNA CASC20 in heterotopic bone formation
Heterotopic Ossification (HO) is a lamellar bone formation in the soft tissues and usually follows injury, trauma or joint replacement. A genome wide association study of HO patients after total hip arthroplasty identified lncRNA Cancer Susceptibility 20 (CASC20) as a gene that is strongly associated with HO severity. Previous findings from my MRes demonstrated an unreported upregulation of CASC20 during BMP2-induced osteodifferentiation of hMSCs. Based on these studies, I hypothesised that CASC20 is a novel regulator of bone formation.
This doctoral thesis investigated the role of CASC20 in regulating osteogenic and chondrogenic differentiation processes. Using a diverse range of experimental models, including hMADs, ASC52teloSOX9, and P512MSCs, this research explored CASC20's impact on key osteogenic and chondrogenic markers. Techniques such as in silico miR prediction, CASC20 lentiviral expression, RT-qPCR, and miR-Seq were used to assess the effect of CASC20 on microRNAs (miRs), genes and biological pathways.
Here I found that CASC20 consistently played a pivotal role in the early stages of osteogenesis and chondrogenesis. CASC20's modulation of miRs emerged as a consistent regulatory mechanism throughout the experiments. The study identified 13 genes commonly targeted by the putative CASC20-interacting miRs, including well-known osteogenic and chondrogenic players such as MAPK1.
To confirm the findings, future steps include genotyping human mesenchymal stem cells and conducting comprehensive experiments to validate the observed effects. The creation of human CRISPR CASC20 KO models will further elucidate CASC20's role. In conclusion, this doctoral thesis provides valuable insights into the intricate mechanisms governed by CASC20 in osteogenesis and chondrogenesis. Further research and exploration are needed to comprehensively elucidate the extent of CASC20's influence in these critical biological processes
Molecular Mechanisms and Therapies of Colorectal Cancer
Colorectal cancer (CRC) is currently the third leading cause of cancer-related mortality, with 1.9 million incidence cases and 0.9 million deaths worldwide. The global number of new CRC cases is predicted to reach 3.2 million in 2040, based on the projection of aging, population growth, and human development.In clinics, despite advances of diagnosis and surgical procedures, 20% of the patients with CRC present with metastasis at the time of diagnosis, caused by residual tumor cells that have spread to distant organs prior to surgery, affecting the patient survival rate. Standard systemic chemotherapy, alternative therapies that target mechanisms involved in cancer progression and metastasis, immunotherapy, and combination therapies are the major CRC-treatment strategies. In the advanced stage of CRC the transforming growth factor-beta (TGF-β) plays an oncogenic role by promoting cancer cell proliferation, cancer cell self-renewal, epithelial-to-mesenchymal transition, invasion, tumor progression, metastatic spread, and immune escape. Furthermore, high levels of TGF-β1 confers poor prognosis and is associated with early recurrence after surgery, resistance to chemo- or immunotherapy, and shorter survival. Based on the body of experimental evidence indicating that TGF-β signaling has the potential to be a good therapeutic target in CRC, several anti-TGF-β drugs have been investigated in cancer clinical trials. Here, we presented a comprehensive collection of manuscripts regarding studies on targeting the TGF-β signaling in CRC to improve patient’s prognosis and personalized treatments
The different functions of sEV-derived miR-574-5p in PGE2-dependent tumors correlate with the tetraspanin composition on the sEV envelope
Small extracellular vesicles (sEV) are essential for intracellular communication in the tumor microenvironment (TME). They can transport biological molecules, such as proteins or nucleic acids, including microRNAs (miRs), a class of short non-coding RNAs. MiRs can exert various functions in gene regulation of target cells. This work aimed to elucidate the transferability of the diverse functions of miR-574-5p to different tumor models. For this purpose, the two prostaglandin E2 (PGE2)-dependent tumor types, non-small cell lung cancer (NSCLC) and neuroblastoma (NB), were investigated. In NSCLC, miR-574-5p regulates the microsomal prostaglandin E2 synthase 1 (mPGES-1)-dependent PGE2 synthesis, which contributes to tumor progression. At the intracellular level, miR-574-5p binds to the CUG-RNA binding protein 1 (CUGBP1), thereby upregulating the synthesis of mPGES-1 and its enzymatic product PGE2. In this thesis, this function of intracellular miR-574-5p was also shown in NB with 11q deletion. Furthermore, NB cells were shown to specifically secrete miR-574-5p into their sEV upon stimulation with PGE2, similar to NSCLC cells. The autocrine role of sEV-derived miR-574-5p in mPGES-1 regulation was not confirmed in NB. However, sEV-miR-574-5p derived from NB cells was shown to exert a novel paracrine role by specific upregulation of the differentiation marker α-smooth muscle actin (α-SMA) of fibroblasts in the TME via Toll-like receptors (TLR) 7/8. Another point investigated in this study was the influence of the tetraspanins CD9, CD63, and CD81 on the functional transfer of sEV-miR-574-5p. In both tumor types, NB and NSCLC, inhibition of specific tetraspanins was shown to alter the function of sEV-miR-574-5p. A difference in the rate of internalization of sEV was excluded as the cause of this functional change. The link between tetraspanins and the functional mediation of sEV-derived miRs is a novel and promising aspect of cancer research.
Overall, this work revealed a new paracrine function of sEV-miR-574-5p, regulating the α-SMA levels of fibroblasts in the TME. Furthermore, the transferability of intracellular and extracellular miR-574-5p functions to different tumor models were analyzed. The interaction of miR-574-5p and CUGBP1 was shown to be transferable from NSCLC to NB, whereas the role of sEV-derived miR-574-5p differed. Investigating the transferability of miR functions to different tumor types is an important approach to maximize the therapeutic benefit of miRs with the least effort and the greatest impact
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The role of microRNAs in skin development
Skin development and hair follicle (HF) cycling are governed by diverse programs of gene modulation through activation and silencing. Post-transcriptional gene expression modulation by microRNAs are another regulatory mechanism to maintain normal skin and HF development and cycling. Here, we identify that miR-148a plays an important role in the regulation of skin homeostasis and HF cycling. RNA and protein analysis of miR-148a and its targets were analysed using both in vitro and in vivo experiments. Spatiotemporal expression analysis of miR-148a during hair cycling revealed an increase in miR-148a expression during the telogen stage, in the bulge and hair germ stem/progenitor cell compartments, and the differentiated, suprabasal layers of the epidermis. We demonstrate that miR-148a can control proliferation and differentiation programs in primary mouse epidermal keratinocytes. Administration of anti-sense miR-148a inhibitor into dorsal mouse skin during telogen stage of HF cycling accelerated telogen-anagen transition and altered stem cell activity, in vitro and in vivo. RNA and protein analysis revealed that miR-148a may control these cellular processes, in part, through modulation of Rock1 and Elf5. This data identifies a previously unknown role of miR-148a and novel gene targets in the control of skin and HF homeostasis and modulating stem/progenitor cell populations. This provides a potential target for stem cell biology research and therapeutic targets for the treatment of skin disorders
RNA, the Epicenter of Genetic Information
The origin story and emergence of molecular biology is muddled. The early triumphs in bacterial genetics and the complexity of animal and plant genomes complicate an intricate history. This book documents the many advances, as well as the prejudices and founder fallacies. It highlights the premature relegation of RNA to simply an intermediate between gene and protein, the underestimation of the amount of information required to program the development of multicellular organisms, and the dawning realization that RNA is the cornerstone of cell biology, development, brain function and probably evolution itself. Key personalities, their hubris as well as prescient predictions are richly illustrated with quotes, archival material, photographs, diagrams and references to bring the people, ideas and discoveries to life, from the conceptual cradles of molecular biology to the current revolution in the understanding of genetic information. Key Features Documents the confused early history of DNA, RNA and proteins - a transformative history of molecular biology like no other. Integrates the influences of biochemistry and genetics on the landscape of molecular biology. Chronicles the important discoveries, preconceptions and misconceptions that retarded or misdirected progress. Highlights major pioneers and contributors to molecular biology, with a focus on RNA and noncoding DNA. Summarizes the mounting evidence for the central roles of non-protein-coding RNA in cell and developmental biology. Provides a thought-provoking retrospective and forward-looking perspective for advanced students and professional researchers
Pig Genomics and Genetics
In this Special Issue, we present the state of the art in the field of pig genetics and genomics, including the identification of gene candidates linked to important pig traits and to nutritional modifications, with the aim of collecting the most recent advances. The published manuscripts focused on high-throughput methodologies, such as RNA sequencing, ATAC-seq, MACE-seq, chip-seq, and RRBS, and covered other fields of pig genetics. The pig (Sus scrofa) is the most common large mammal in the world. The Sus genus includes domestic pig and wild boar. Since the draft reference genome sequence of S. scrofa was assembled in 2012, the processes of identification of genes related to important phenotypic traits and of search of genetic markers for pig selection have been significantly refined. In addition, the newest wide-range high-throughput techniques, including microarrays, next-generation sequencing, and the recent PacBio sequencing platform providing ultra-long sequencing reads, allow identifying gene mutations and gene candidates throughout the whole genome, transcriptome, or epigenome and estimating quantitative traits important for breeding as well as the genetic backgrounds of inherited diseases
Evaluation of blood-based microRNAs toward clinical use as biomarkers in common and rare diseases
According to the GLOBOCAN project of the International Agency for Research on Cancer, the top three common cancer diseases worldwide in the year 2020 were breast, lung and colorectal cancer. These are usually diagnosed via imaging methods (e.g. computer tomography) or invasive methods (e.g. biopsy). However, these techniques are potentially risky and expensive and thus not accessible to all patients, resulting in most cancers being detected in an advanced stage. Since the discovery of small non-coding RNAs and specifically microRNAs and their role as gene regulators, many researchers investigate their association with disease development. In particular, researchers examine body fluid based microRNAs which could present potential cost-effective and minimally- or non-invasive alternatives to the previously described established diagnosis methods.
This dissertation focuses on microRNAs and investigates their suitability as minimally-invasive blood-borne biomarkers for potential diagnostic purposes. More specifically, the goals of this work are (1) to implement a new method to predict novel microRNAs, (2) to understand stability and characteristics of these small non-coding RNAs, possibly relevant for the last goal, (3) to discover potential diagnostic biomarkers in common and rare diseases. The first goal was addressed by developing miRMaster, a web service to predict new microRNAs. The tool uses machine learning and high-throughput sequencing data to find microRNA candidates that follow the known biogenesis pathways. The second goal was pursued in four publications. First, we performed a large scale evaluation of miRMaster by generating a high-resolution map of the human small non-coding RNA transcriptome for which we analyzed and validated potential microRNA candidates. Next, we examined the influence of seasonal effects on microRNA expression profiles and observed the largest difference between spring and the other seasons. Additionally, we evaluated the evolutionary conservation of small non-coding RNAs in zoo animals and showed that the distribution of sncRNA classes varies across species, while common microRNA families are present in more diverse organisms than assumed so far. Furthermore, we analyzed if microRNAs are technically stable, and whether biological variation is preserved when using capillary dried blood spots as an alternative sample collection device to venous blood specimens. Finally, we investigated the suitability of microRNAs as biomarkers for two diseases: lung cancer and Marfan disease. We identified blood-borne biomarker candidates for lung cancer detection in a large-scale multi-center study via machine learning. For the rare Marfan disease we analyzed the paired messenger RNA and microRNA expression levels in whole-blood samples. This highlighted several significantly deregulated microRNAs and messenger RNAs, which we subsequently validated in an independent cohort.
In summary, this thesis provides valuable results toward potential clinical use of microRNAs, and the herein described projects represent comprehensive analyses of them from different perspectives: starting with microRNA discovery, addressing various technical and biological questions and ending with the potential use as biomarkers.Nach Angaben des GLOBOCAN-Projekts der International Agency
for Research on Cancer sind die drei häufigsten Krebserkrankungen
weltweit im Jahr 2020 Brust-, Lungen- und Darmkrebs. Diese werden in
der Regel durch bildgebende Verfahren (z.B. Computertomographie)
oder invasive Methoden (z.B. Biopsie) diagnostiziert. Diese Verfahren
sind jedoch potenziell risikoreich und teuer und daher nicht für alle
Patienten zugänglich. Dies führt dazu, dass die meisten Krebsarten
erst in einem fortgeschrittenen Stadium entdeckt werden. Seit der
Entdeckung der kurzen nichtkodierenden RNAs und insbesondere
der microRNAs und ihrer Rolle als Genregulatoren untersuchen viele
Forscher ihren Zusammenhang mit der Krankheitsentwicklung. Insbesondere
untersuchen die Forscher die in Körperflüssigkeiten vorkommenden
microRNAs, die potenziell kosteneffiziente und minimal- oder
nicht-invasive Alternativen zu den bisher beschriebenen etablierten
Diagnosemethoden darstellen könnten.
Diese Dissertation konzentriert sich auf microRNAs und untersucht
deren Eignung als minimal-invasive blutbasierte Biomarker
für potenzielle diagnostische Zwecke. Genauer gesagt sind die Ziele
dieser Arbeit (1) die Implementierung einer neuen Methode zur
Vorhersage neuartiger microRNAs, (2) das Verständnis über die Stabilität
und Charakteristika dieser kurzen nicht-kodierenden RNAs, die
möglicherweise für das nächste Ziel relevant sind, (3) die Entdeckung
potenzieller diagnostischer Biomarker für verschiedene Anwendungen.
Das erste Ziel wurde durch die Entwicklung von miRMaster verfolgt,
einem Webdienst zur Vorhersage neuer microRNAs. Das Tool nutzt
maschinelles Lernen und Hochdurchsatz-Sequenzierungsdaten, um
microRNA-Kandidaten zu finden, die den bekannten Wege der
Biogenese folgen. Das zweite Ziel wurde in vier Veröffentlichungen
verfolgt. Zunächst führten wir eine groß angelegte Evaluierung
von miRMaster durch, indem wir eine High-Resolution Map des
menschlichen Transkriptoms kurzer nichtkodierender RNAs erstellten,
für die wir potenzielle microRNA-Kandidaten analysierten und
validierten. Anschließend untersuchten wir den Einfluss saisonaler
Effekte auf die microRNA-Expressionsprofile und beobachteten
den größten Unterschied zwischen dem Frühling und den anderen
Jahreszeiten. Darüber hinaus untersuchten wir die evolutionäre
Erhaltung kurzer nichtkodierender RNAs in Zoo-Tieren und zeigten,
dass die Verteilung der kurzer nichtkodierenden RNA-Klassen zwischen
den Arten variiert, während gemeinsame microRNA-Familien
in verschiedeneren Organismen vorkommen als bisher angenommen.
Darüber hinaus analysierten wir, ob microRNAs technisch
stabil sind und ob die biologische Variation erhalten bleibt, wenn
kapillares Trockenblut als alternatives Probenentnahmeverfahren zu
venösen Blutproben verwendet werden. Schließlich untersuchten wir
die Eignung von microRNAs als Biomarker für zwei Krankheiten:
Lungenkrebs und Marfan-Krankheit. In einer groß angelegten multizentrischen
Studie identifizierten wir mit Hilfe von maschinellem
Lernen Biomarker-Kandidaten aus dem Blut für die Erkennung von
Lungenkrebs. Für die seltene Marfan-Krankheit analysierten wir die
gepaarten Expressionsniveaus von messengerRNA und microRNA
in Vollblutproben. Dabei wurden mehrere signifikant deregulierte
microRNAs und messengerRNAs festgestellt, die wir anschließend in
einer unabhängigen Kohorte validierten.
Zusammenfassend lässt sich sagen, dass diese Arbeit wertvolle
Ergebnisse im Hinblick auf die potenzielle klinische Verwendung von
microRNAs liefert. Die hier beschriebenen Projekte stellen umfassende
Analysen aus verschiedenen Blickwinkeln dar: angefangen bei der
Entdeckung von microRNAs, über verschiedene technische und biologische
Fragen bis hin zur potenziellen Verwendung als Biomarker
Role of miRNAs in Cancer
MicroRNAs are the best representatives of the non-coding part of the genome and their functions are mostly linked to their target genes. During the process of carcinogenesis, both dysregulation of microRNAs and their target genes can explain the development of the disease. However, most of the target genes of microRNAs have not yet been elucidated. In this book, we add new information related to the functions of microRNAs in various tumors and their associated targetome
Cutaneous Melanoma Classification: The Importance of High-Throughput Genomic Technologies
Cutaneous melanoma is an aggressive tumor responsible for 90% of mortality related to
skin cancer. In the recent years, the discovery of driving mutations in melanoma has led to
better treatment approaches. The last decade has seen a genomic revolution in the field of
cancer. Such genomic revolution has led to the production of an unprecedented mole of
data. High-throughput genomic technologies have facilitated the genomic, transcriptomic
and epigenomic profiling of several cancers, including melanoma. Nevertheless, there are
a number of newer genomic technologies that have not yet been employed in large
studies. In this article we describe the current classification of cutaneous melanoma, we
review the current knowledge of the main genetic alterations of cutaneous melanoma and
their related impact on targeted therapies, and we describe the most recent highthroughput genomic technologies, highlighting their advantages and disadvantages. We
hope that the current review will also help scientists to identify the most suitable
technology to address melanoma-related relevant questions. The translation of this
knowledge and all actual advancements into the clinical practice will be helpful in
better defining the different molecular subsets of melanoma patients and provide new
tools to address relevant questions on disease management. Genomic technologies
might indeed allow to better predict the biological - and, subsequently, clinical - behavior
for each subset of melanoma patients as well as to even identify all molecular changes in
tumor cell populations during disease evolution toward a real achievement of a
personalized medicine
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