An Integrated Regulatory Network Based on Comprehensive Analysis of mRNA Expression, Gene Methylation and Expression of Long Non-coding RNAs (lncRNAs) in Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are a heterogeneous group of disorders characterized by ineffective hematopoiesis, defective differentiation of hematopoietic precursors, and expansion of the abnormal clones. The prevalence of MDS has raised great concerns worldwide, but its pathogenetic mechanisms remain elusive. To provide insights on novel biomarkers for the diagnosis and therapy of MDS, we performed high-throughput genome-wide mRNA expression profiling, DNA methylation analysis, and long non-coding RNAs (lncRNA) analysis on bone marrows from four MDS patients and four age-matched healthy controls. We identified 1,937 differentially expressed genes (DEGs), 515 methylated genes, and 214 lncRNA that showed statistically significant differences. As the most significant module-related DEGs, TCL1A, PTGS2, and MME were revealed to be enriched in regulation of cell differentiation and cell death pathways. In addition, the GeneGo pathway maps identified by top DEGs were shown to converge on cancer, immunoregulation, apoptosis and regulation of actin cytoskeleton, most of which are known contributors in MDS etiology and pathogenesis. Notably, as potential biomarkers for diagnosis of MDS, four specific genes (ABAT, FADD, DAPP1, and SMPD3) were further subjected to detailed pathway analysis. Our integrative analysis on mRNA expression, gene methylation and lncRNAs profiling facilitates further understanding of the pathogenesis of MDS, and may promote the diagnosis and novel therapeutics for this disease

The Role of Long Noncoding RNAs in Gene Expression Regulation

Accumulating evidence highlights that noncoding RNAs, especially the long noncoding RNAs (lncRNAs), are critical regulators of gene expression in development, differentiation, and human diseases, such as cancers and heart diseases. The regulatory mechanisms of lncRNAs have been categorized into four major archetypes: signals, decoys, scaffolds, and guides. Increasing evidence points that lncRNAs are able to regulate almost every cellular process by their binding to proteins, mRNAs, miRNA, and/or DNAs. In this review, we present the recent research advances about the regulatory mechanisms of lncRNA in gene expression at various levels, including pretranscription, transcription regulation, and posttranscription regulation. We also introduce the interaction between lncRNA and DNA, RNA and protein, and the bioinformatics applications on lncRNA research

Včasná detekce progrese onemocnění u pacientů s myelodysplastickým syndromem.

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic disorders with a risk of transformation into acute myeloid leukemia (AML). The International Prognostic Scoring Systems integrate clinical data and cytogenetics to determine the risk of AML transformation for individual patients. Precise risk assessment is crucial for treatment decision- making. The aim of this thesis was to identify molecular markers for the early detection of disease progression in MDS patients. Using cDNA microarrays and next-generation sequencing, we targeted long noncoding RNAs (lncRNAs) and recurrently mutated genes in bone marrow cells. In addition, we focused on the identification of pathways related to the progression of MDS and understanding how the identified biomarkers participate. In the transcriptome study, we identify 4 candidate lncRNAs that may serve as prognostic biomarkers of the adverse course of MDS: H19, WT1-AS, TCL6, and LEF1-AS. Using various statistical approaches, we determined the level of H19 to be a strong independent prognostic marker. Furthermore, our data showed that disruption of transcriptional coregulation of the imprinting locus H19/IGF2 and miR-675, which directly regulates H19 and plays a role in tumorigenesis, accompanies disease progression. In the genomic study...Myelodysplastický syndrom (MDS) je heterogenní skupina onemocnění charakterizována klonální poruchou krvetvorby s rizikem transformace do akutní myeloidní leukémie (AML). Na základě vyšetření krevního obrazu, kostní dřeně a cytogenetiky je podle mezinárodních prognostických skórovacích systémů určováno riziko transformace do AML. Přesné určení rizika je klíčové pro zvolení správné léčby. Cílem této práce byla identifikace molekulárních markerů pro včasnou detekci progrese onemocnění. Pomocí cDNA čipů a sekvenování nové generace byly analyzovány dlouhé nekódující RNA (lncRNA) a rekurentně mutované geny v buňkách kostní dřeně. Zároveň bylo naším cílem popsání signálních drah, které se podílí na progresi onemocnění, a vysvětlení, jak dané biomarkery k progresi přispívají. V transkriptomové studii jsme identifikovali 4 kandidátní lncRNA, které by mohly sloužit jako prognostické biomarkery horšího průběhu MDS, a to H19, WT1-AS, TCL6 a LEF1-AS1. Na základě několika statistických přístupů jsme prokázali, že hladina transkriptu H19 může sloužit jako velmi silný nezávislý prognostický marker. Navíc naše data ukázala, že progrese je doprovázena poruchou transkripční regulace imprintovaného lokusu H19/IGF2 a miR-675, která přímo reguluje H19 a hraje významnou roli v tumorigenezi. V genomické studii zaměřené...Ústav klinické a experimentální hematologie 1. LF UK a ÚHKTInstitute of Clinical and Experimental Hematology First Faculty of Medicine Charles University and Institute of Hematology and Blood TransfusionFirst Faculty of Medicine1. lékařská fakult

LncRNAs signature defining major subtypes of B-cell acute lymphoblastic leukemia

Introduction: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most prevalent heterogeneous cancer in children and adults, with multiple subtypes. Emerging evidence suggests that long non-coding RNAs (lncRNAs) might play a key role in the development and progression of leukemia. Thus, we performed a transcriptional and DNA methylation survey to explore the lncRNA landscape on three BCP-ALL subtypes (82 samples) and demonstrated their functions and epigenetic profile. Methodology: The primary BCP-ALL samples from bone marrow material were collected from diagnosis (ID) and relapse (REL) stages of adult (n = 21) and pediatric (n = 24) BCP-ALL patients, using RNA-seq and DNA methylation array technology. The subtype-specific and relapse-specific lncRNAs were analyzed by differential expression (DE) analysis method using LIMMA Voom. By analyzing the co-expression of the subtype-specific lncRNAs and protein-coding (PC) genes from all subtypes, we inferred potential functions of these lncRNAs by applying “guilt-by-association” approach. Additionally, we validated our subtype-specific lncRNAs on an independent cohort of 47 BCP-ALL samples. The epigenetic regulation of subtype-specific lncRNAs were identified using the Bumphunter package. The correlation analysis was performed between DM and DE lncRNAs from three subtypes to determine the epigenetically facilitated and silenced lncRNAs. Results: We present a comprehensive landscape of lncRNAs signatures which classifies three molecular subtypes of BCP-ALL on DNA methylation and RNA expression levels. The principle component analysis (PCA) on most variable lncRNAs on RNA and DNA methylation level confirmed robust separation of DUX4, Ph-like and NH-HeH BCP-ALL subtypes. Using integrative bioinformatics analysis, subtype-specific and relapse-specific lncRNAs signature together determine 1564 subtype-specific and 941 relapse-specific lncRNAs from three subtypes. The unsupervised hierarchical clustering on these subtype-specific lncRNAs validated their specificity on the independent validation cohort. For the first time, our study demonstrates that BCP-ALL subtype specific as well as relapse-specific lncRNAs may contribute to the activation of key pathways including TGF-β, PI3K-Akt, mTOR and activation of JAK-STAT signaling pathways from DUX4 and Ph-like subtypes. Finally, the significantly hyper-methylated and hypo-methylated subtype-specific lncRNAs were profiled. In addition to that, we identified 23 subtypes specific lncRNAs showing hypo and hyper-methylation pattern in their promoter region that significantly correlates with their diminished and increased expression in respective subtypes. Conclusions: Overall, our work provides the most comprehensive analyses for lncRNAs in BCP-ALL subtypes. Our findings suggest a wide range of biological functions associated with lncRNAs and epigenetically facilitated lncRNAs in BCP-ALL and provide a foundation for functional investigations that could lead to novel therapeutic approaches.Einführung: Die B-Vorläufer akute lymphatischen Leukämie (BCP-ALL) ist eine heterogene Krebserkrankung mit mehreren definierten Subgruppen. Neue Daten deuten darauf hin, dass lange nicht-kodierende RNAs (long noncoding RNAs - lncRNAs) eine Schlüsselrolle bei der Entwicklung und Progression der BCP-ALL spielen könnten. Daher führten wir eine Transkriptions- und DNA-Methylierungsstudie durch, um die lncRNA-Landschaft von drei BCP-ALL-Subgruppen (82 Proben) zu charakterisieren und potentielle regulative Konsequenzen zu analysieren. Methodik: Material wurde zum Zeitpunkt der Erstdiagnose (ID) und im Rezidiv (REL) von erwachenen (n = 21) und pädiatrischen (n = 24) BCP-ALL-Patienten entnommen und unter Verwendung von RNA-Seq und DNA-Methylierungs-Array-Technologien untersucht. Die Subgruppen-spezifischen und rezidiv-spezifischen lncRNAs wurden durch differentielle Expressions (DE) Analysen mit LIMMA Voom analysiert. Durch die Analyse der Koexpression von lncRNAs mit Protein-kodierenden (PC) Genen aus allen Subgruppen schlossen wir unter Verwendung eines ‚Guilt-by-association‘ -Ansatzes auf potentielle Funktionen der DE lncRNAs. Zudem haben wir die Subgruppen-spezifischen lncRNAs auf einem unabhängigen Datenset von 47 BCP-ALL-Proben validiert. Die epigenetische. Die epigenetische Regulation von Subgruppen-spezifischen lncRNAs wurde durch eine differentielle Methylierungs (DM) analyse identifiziert. Die Korrelation zwischen DM und DE lncRNAs aus drei Subgruppen wurde ermittelt, um den Einfluss der epigenetischen Regulation auf die Expression von lncRNAs zu analysieren. Ergebnisse: Wir präsentieren eine umfassende Landschaft von lncRNA-Signaturen, die drei molekulare Subtypen von BCP-ALL auf DNA-Methylierungs- und RNA-Expressionslevel klassifiziert. Die Hauptkomponentenanalyse (PCA) auf den top variablen lncRNAs auf RNA und DNA-Methylierungsniveau bestätigte eine robuste Trennung von Ph-like, DUX4 und NH-NeH BCP-ALL Subtypen. Mit integrativer bioinformatischer Analyse, zusammen 1564 subtyp-spezifische und 941 rezidiv-spezifische lncRNAs aus den drei Subtypen. Das unüberwachte hierarchische Clustering auf diesen Subtyp-spezifischen lncRNAs validierte ihre Spezifität in der unabhängigen Validierungskohorte. Unsere Studie zeigt erstmals, dass BCP-ALL-Subtyp-spezifische sowie Rezidiv-spezifische lncRNAs zur Aktivierung von Signalwegen wie TGF-β, PI3K-Akt, mTOR und Aktivierung von JAK-STAT-Signalwegen von DUX4 und Ph-like Subtypen. Endlich wurden die signifikant DM subtyp-spezifische lncRNAs profiliert. Darüber hinaus identifizierten wir 23 Subtyp-spezifische lncRNAs, die ein Hypo- und Hypermethylierungsmuster in ihrer Promotorregion zeigen, das signifikant mit ihrer verringerten und erhöhten Expression in den jeweiligen Subtypen korreliert. Schlussfolgerungen: Insgesamt liefert unsere Arbeit die umfassendsten Analysen für lncRNAs in BCP-ALL-Subtypen. Unsere Ergebnisse weisen auf eine Vielzahl von biologischen Funktionen im Zusammenhang mit lncRNAs und epigenetisch erleichterten lncRNAs in BCP-ALL hin und bieten eine Grundlage für funktionelle Untersuchungen, die zu neuen therapeutischen Ansätzen führen könnten

Gene expression studies from basic research to the clinic

Humane genetica is een spannend en multidisciplinair vakgebied. De focus van onderzoek binnen de humane genetica ligt op erfelijke ziekten, en het doel van het onderzoek is het verlichten van lijden door het behandelen en voorkomen van diverse ziekten.Het grootste project in de biologie tot dusver, het Humane Genoomproject (the Human Genome Project), is in 2003 afgerond en onthulde voor het eerst de hele DNA-sequentie van het menselijke genoom. Sindsdien zijn de kosten van en tijd die nodig is voor sequencing en genotypering van menselijke genen drastisch gedaald. Dit maakt grote studies van honderden duizenden individuen, en in de nabije toekomst, van miljoenen individuen, mogelijk.Als gevolg van deze grootschalige genetische studies, realiseren we ons voor het eerst hoe complex de genetica van de mens eigenlijk is. Honderden genetische varianten blijken in genoomwijde associatiestudies met een specifiek fenotype of ziekte geassocieerd. Deze varianten verklaren echter meestal slechts een klein deel van de erfelijkheid van dit fenotype. Bovendien zijn de mechanismen waarmee deze varianten tot ziekte leiden meestal nog steeds onbekend.DNA is een prachtig en veelzijdig molecuul, het bevat informatie waarmee het repliceert en waarmee het organisme zich in zijn omgeving kan ontwikkelen. In cellen worden delen van het DNA (de genen) overgeschreven naar RNA. Sommige RNA-moleculen (van voor eiwit coderende genen) worden verder omgezet naar eiwitten (translatie). Andere RNA-moleculen spelen uiteenlopende rollen, zoals bijvoorbeeld het reguleren van de transcriptie van andere genen.RNA transcriptie (ook wel genexpressie genoemd) is de eerste stap in het process van DNA naar fenotype. Daarom kan het bestuderen van kwantiteit aan RNA in monsters uit verschillende weefsels ons helpen bij het begrijpen van cellulaire fenomenen die tot ziekte leiden.In dit proefschrift heb ik grote hoeveelheden publiek beschikbare genexpressiegegevens gebruikt om de functies van genen te voorspellen en genen en de gen-specifieke netwerken (in het Engels: pathways) te prioriteren die relevant kunnen zijn voor verschillende fenotypen en ziekten.Human genetics is an exciting, multidisciplinary field of science. Its focus of investigation is on hereditary diseases in humans, with the ultimate aim of alleviating suffering by treating and preventing various diseases.The biggest project in biology to date, the Human Genome Project, was completed in 2003 and it unravelled the DNA sequence of the human genome. Since then, the cost and time needed for sequencing and genotyping human genomes have fallen dramatically, which now allows for massive studies to be performed consisting of hundreds of thousands of individuals. In the near future, studies of millions of people are likely to appear.As a result of the large-scale studies during the past decade, we have come to appreciate the true complexity of genetics. In genome-wide association studies, hundreds of genetic variants have been associated with a specific phenotype or disease. However, these variants together typically only explain a small proportion of the heritability of the phenotype, while the mechanisms by which such variants lead to disease are still mostly unknown.DNA is a marvellous molecule. It contains information which enables it to replicate itself and make the organism develop in its environment. In cells, parts of DNA (i.e. genes) are transcribed into RNA. Some RNA molecules (protein-coding genes) are further translated into proteins. Other RNAs play different roles, such as regulating the transcription of other genes.RNA transcription (gene expression) is the first step in the DNA-to-phenotype process. Therefore, studying abundances of RNA in samples from various tissues can help us to un- derstand the cellular phenomena that lead to disease.In this thesis, I report on my research using publicly available gene expression data on a large scale to predict the functions of genes and to prioritize genes and pathways that may be relevant to different phenotypes and diseases

microRNAs in Cancer: biological effects of microRNAs in colorectal cancer

Tese de Doutoramento em Ciências da SaúdeNon-coding RNAs are a class of RNA transcripts that do not codify for protein. Included in this class are the microRNAs, small RNAs with approximately 20 nucleotides in length. microRNAs act post-transcriptionally by binding to 5´- ultraconserved regions, coding regions or 3´-ultraconserved regions of messenger RNAs and inhibit its translation or cause messenger RNA cleavage. Additionally, microRNAs can also bind to pseudogenes and are involved in RNA decoy functions. A single microRNA can bind to several RNAs and a single RNA can be targeted by different microRNAs. Therefore, microRNAs constitute master regulators of gene expression and major key players in the control of cellular functions. microRNAs are deregulated in all tumor types and can act as tumor suppressors or as oncogenes in a tissue- and cell-specific manner. microRNAs control biological mechanisms essential for cancer cells, such as proliferation, cell death, cell cycle, metabolism, hypoxia, angiogenesis, inflammation, migration, invasion, metastasis and cancer stem cells self-renewal. Besides microRNAs´ importance as diagnostic and prognostic tools in cancer, and due to their involvement in cancer biology, microRNAs have been regarded as new therapeutic targets. This thesis focuses on the role of microRNAs on colorectal cancer. Despite the great improvement in therapeutics, colorectal cancer is still one of the most commonly diagnosed tumor types and a frequent cause of cancer-related death. We intend to understand the biological effects of miR-28 in this tumor type. To achieve this goal both in vitro and in vivo approaches were used. In the microRNA biogenesis pathway, usually a primary-microRNA is processed into a precursor-microRNA, which originates a single mature microRNA. However, in some cases, two mature miRNAs are processed. This occurs with the precursor-miR-28 that is processed into two mature microRNAs that were named miR-28-5p and miR-28- 3p. We identified for the first time that both mature forms of miR-28 were deregulated in colorectal cancer. Contrary to some tumor and cell types in which miR-28 was found to be upregulated, in colorectal cancer miR-28 is downregulated in tumor compared with normal colorectal tissue. However, the two mature forms have distinct functions. Our in vitro results revealed that miR-28-5p overexpression decreased proliferation, increased apoptosis, caused a G1-arrest in the cell cycle, and decreased migration and invasion, while miR-28-3p overexpression had no effect on proliferation but increased migration and invasion. This was the first description of miR-28 dual role in colorectal cancer cells. As miR-28 mature forms are transcribed together, we analyzed the overall result in vivo. Xenografts of colorectal cancer cells overexpressing miR-28 caused a slower tumor growth in mice, while increased metastasis, when compared with the control group. In this thesis we also discuss the strategies to inhibit or restore microRNAs function. The dual role of miR-28 has great impact on the design of therapies using microRNAs, in particular for upregulation of microRNA levels using expression vectors. Variation in expression levels of microRNAs has a tremendous impact on the intracellular signaling networks. We determined that miR-28-5p targets Cyclin D1, an important regulator of cell cycle progression, in particular of the G1-S phase transition. Additionally, miR-28-5p directly targets HOXB3, a transcription factor described to be upregulated in colon cancer. On the other hand, miR-28-3p directly regulates the expression of NM23-H1, an anti-metastatic gene. miR-28 is part of an extensive number of microRNAs deregulated in colorectal cancer. It is essential to understand the effect of the microRNAs in cancer to be able to interfere with the tumor biology and consequently generate therapeutic strategies based on microRNAs as an approach to fight cancer.RNAs não codificantes são uma classe de transcritos de RNA que não codificam proteína. Dentro desta classe encontram-se os microRNAs, pequenos RNAs com 20 nucleótidos de comprimento. Os microRNAs actuam após a transcrição e ligam-se por complementariedade de bases a RNAs mensageiros, nas suas regiões 5´- ou 3´-ultraconversadas e nas regiões codificantes. Os microRNAs inibem a tradução ou causam a degradação do RNA mensageiro. Além disso, os microRNAs podem também ligar-se a pseudo-genes e estar envolvidos em funções de RNA “decoy”. Um único microRNA pode ligar-se a vários RNAs, e um único RNA pode ser o alvo de vários microRNAs. Assim sendo, os microRNAs constituem excelentes reguladores da expressão genética e são elementos chave no controlo das funções celulares. Os microRNAs estão desregulados em todos os tipos de tumor e podem actuar como supressores tumorais ou como oncogenes, dependendo do tipo de tecido e de célula. Os microRNAs controlam mecanismos biológicos essenciais para as células neoplásicas, incluindo a proliferação, a morte e o ciclo celular, o metabolismo, a hipoxia, a angiogénese, a inflamação, a migração, a invasão, a metastização e a autorenovação das células estaminais tumorais. Para além da importância dos microRNAs como ferramentas de diagnóstico e prognóstico oncológico, e devido ao seu envolvimento na biologia tumoral, os microRNAs têm sido apresentados como novos alvos terapêuticos. Esta tese analisa o papel dos microRNAs no cancro colorectal. Apesar dos significativos avanços terapêuticos, o cancro colorectal continua a ser um dos tipos tumorais mais diagnosticados e uma frequente causa de morte por cancro. No trabalho aqui apresentado, pretende-se analisar o efeito biológico do miR-28 neste tipo tumoral. Para atingir este objectivo utilizámos ferramentas in vitro e in vivo.Geralmente, na via da biogénese dos microRNAs, um microRNA-primário é processado constituindo o microRNA-precursor que, por sua vez, origina um único microRNA maduro. No entanto, em alguns casos, são originados dois microRNAs maduros. Esta situação ocorre com o precursor do miR-28 que é processado em dois microRNAs maduros designados de miR-28-5p e miR-28-3p. Identificámos, pela primeira vez, ambas as formas do miR-28 maduro como estando desreguladas em cancro colorectal. Contrariamente a outros tipos de tumores/células nas quais o miR- 28 está sobreexpresso, no cancro colorectal o miR-28 está subexpresso no tumor quando comparado com os seus níveis de expressão no tecido colorectal normal. No entanto, as duas formas maduras têm funções distintas. Os estudos in vitro revelaram que a sobreexpressão do miR-28-5p diminui a proliferação, aumenta a apoptose, impede a progressão do ciclo celular na fase G1, e diminui a migração e a invasão; enquanto que a sobreexpressão do miR-28-3p não tem efeito na proliferação mas aumenta a migração e a invasão. Esta é a primeira descrição do duplo papel do miR-28 em neoplasia colorectal. Visto que as formas maduras do miR-28 são transcritas conjuntamente, analisámos o resultado global da expressão do miR-28 in vivo. A injecção, em murganhos, de células neoplásicas colorectais que sobreexpressam miR- 28 causou um crescimento mais lento dos tumores, mas provocou um aumento no número de metastases, quando comparámos com o grupo control. Nesta tese, discutimos as estratégias para inibir ou restaurar a função dos microRNAs. O duplo papel do miR-28 tem um vasto impacto no desenho de terapias que recorram a microRNAs, em particular na sobreexpressão dos níveis de microRNAs usando vectores de expressão. A variação dos níveis de microRNAs tem um impacto tremendo na rede de sinalização intracellular. Observámos que o miR-28-5p tem como alvo a Cyclin D1, um importante regulador da progressão no ciclo celular, em particular na transição G1-S. Além disso, o miR-28-5p tem como alvo directo o HOXB3, um factor de transcrição descrito como estando sobreexpresso no cancro do colon. Por outro lado, o miR-28-3p regula directamente a expressão de NM23-H1, um gene anti-metastático. O miR-28 faz parte de um extenso número de microRNAs que está desregulado em neoplasias. Compreender o efeito dos microRNAs no cancro é essencial para interferir com a sua biologia e, consequentemente, gerar estratégias terapêuticas utilizando microRNAs na luta contra esta doença devastadora

Gene Expression Profiling in Cancer

The contribution of modern-day genetics in designing efficient gene expression profiles for cancer is immense. The progress of technology and science in recent years provides the opportunity for discovery and application of new techniques for treating various diseases that affect humanity. Methods for finding and analyzing the profile of gene expression of infected cells give scientists the ability to develop more targeted and effective treatments, especially for diseases such as cancer. The development of gene expression profiling is one of the most important achievements in cancer genetics in our time. It is essentially the driving force behind personalized and precision medicine. This book highlights recent developments, applications, and breakthroughs in the field of gene expression profiling in cancer