MicroRNA regulation of CD8+ T cell responses.

MicroRNAs (miRNAs) are a class of short noncoding RNAs that play critical roles in the regulation of a broad range of biological processes. Like transcription factors, miRNAs exert their effects by modulating the expression of networks of genes that operate in common or convergent pathways. CD8+ T cells are critical agents of the adaptive immune system that provide protection from infection and cancer. Here, we review the important roles of miRNAs in the regulation of CD8+ T cell biology and provide perspectives on the broader emerging principles of miRNA function

FUNCTIONAL STUDIES OF microRNAs IN DEVELOPMENT AND CANCER

MicroRNAs (miRNAs) COMPRISE a large family of small (~23 nucleotide in length), endogenous RNAs that regulate gene expression at the posttranscriptional level. Functional studies have indicated that miRNAs participate in the regulation of nearly all cellular processes investigated so far, including differentiation, apoptosis, and proliferation. Further, the deregulation of miRNA expression greatly contributes to human diseases, and is associated with many human pathologies, such as cancer. The studies in this thesis have focused on miRNA expression and regulation in various forms of malignancies. Specifically, we wanted to provide mechanistic insights into the role of miRNAs in tumorigenesis. In parallel, we hoped to discover new therapeutic targets that could be exploited clinically to treat childhood and adult cancer. In the work presented, we describe the functional consequences of miRNA perturbations in three distinct neoplasias: (1) chronic lymphocytic leukemia (CLL), the second most common type of blood cancer in adults; (2) neuroblastoma (NB), an embryonal malignancy of the sympathetic nervous system that is derived from primordial neural crest cells and occurs almost exclusively in infants and young children; and, (3) basal cell carcinoma (BCC), a basal cell-derived malignancy of the epidermis, which ranks as the most commonly diagnosed human cancer among fair-skinned individuals. Our CLL studies revealed that the DLEU2 transcript functions as a regulatory host gene for the miRNAs miR-15a and miR-16-1. These miRNAs were shown to target the G1 cyclins D1 and E1 for translational repression, resulting in a prominent cell cycle arrest. Further, ectopic expression of DLEU2 inhibited the colony-forming capacity of tumor cell lines, suggesting a tumor-suppressive function for miR-15a and miR-16-1. We also demonstrate that DLEU2 is transcriptionally regulated by the oncoprotein c-MYC, providing a novel mechanism by which MYC can regulate the G1 cyclins in a posttranscriptional manner. Functional loss of DLEU2 may thus constitute an important step in CLL tumorigenesis and various c-MYC-dependent cancers. In our analysis of MYCN-amplified neuroblastoma (NB), we investigated the molecular consequences and functional outcome of abnormal miRNA regulation and discovered that miR-17~92 cluster-derived miRNAs potentiate the tumorigenic behavior of this childhood cancer. Importantly, we could show that miR-18a and miR-19a target and repress the expression of estrogen receptor-α (ESR1), a ligand-inducible transcription factor implicated in neuronal differentiation. We propose that ESR1 represents a previously undescribed MYCN target in NB and demonstrate a unique oncogenic circuitry in which the repression of ESR1 through MYCN-regulated miRNAs may play a fundamental role in NB tumorigenesis. Finally, based on our genome-wide miRNA expression analysis of a non-melanoma skin cancer, we found that the skin-specific miRNA, miR-203, is preferentially lost in BCC. Functional analyses demonstrated that the inappropriate activation of the Hedgehog and MAPK pathways in BCCs may contribute to cancer progression via severely reduced expression of miR-203, which dramatically facilitates the misexpression of genes involved in the regulation of cell proliferation and cell cycle, including c-JUN and c-MYC. In this respect, miR-203 constitutes a gatekeeper miRNA controlling keratinocyte proliferation. The molecular reconstitution of miR-203 could therefore serve as a novel therapeutic strategy in the treatment of BCC tumors

SWIM: A computational tool to unveiling crucial nodes in complex biological networks

SWItchMiner (SWIM) is a wizard-like software implementation of a procedure, previously described, able to extract information contained in complex networks. Specifically, SWIM allows unearthing the existence of a new class of hubs, called "fight-club hubs", characterized by a marked negative correlation with their first nearest neighbors. Among them, a special subset of genes, called "switch genes", appears to be characterized by an unusual pattern of intra- and inter-module connections that confers them a crucial topological role, interestingly mirrored by the evidence of their clinic-biological relevance. Here, we applied SWIM to a large panel of cancer datasets from The Cancer Genome Atlas, in order to highlight switch genes that could be critically associated with the drastic changes in the physiological state of cells or tissues induced by the cancer development. We discovered that switch genes are found in all cancers we studied and they encompass protein coding genes and non-coding RNAs, recovering many known key cancer players but also many new potential biomarkers not yet characterized in cancer context. Furthermore, SWIM is amenable to detect switch genes in different organisms and cell conditions, with the potential to uncover important players in biologically relevant scenarios, including but not limited to human cancer

Cancer systems biology: exploring cancer-associated genes on cellular networks

Genomic alterations lead to cancer complexity and form a major hurdle for a comprehensive understanding of the molecular mechanisms underlying oncogenesis. In this review, we describe the recent advances in studying cancer-associated genes from a systems biological point of view. The integration of known cancer genes onto protein and signaling networks reveals the characteristics of cancer genes within networks. This approach shows that cancer genes often function as network hub proteins which are involved in many cellular processes and form focal nodes in the information exchange between many signaling pathways. Literature mining allows constructing gene-gene networks, in which new cancer genes can be identified. The gene expression profiles of cancer cells are used for reconstructing gene regulatory networks. By doing so, the genes, which are involved in the regulation of cancer progression, can be picked up from these networks after which their functions can be further confirmed in the laboratory.Comment: More similar papers at http://www.bri.nrc.ca/wan

Circ Res

Rationale and ObjectiveIn this Emerging Science Review, we discuss a systems genetics strategy, which we call Gene Module Association Study (GMAS), as a novel approach complementing Genome Wide Association Studies (GWAS), to understand complex diseases by focusing on how genes work together in groups rather than singly.MethodsThe first step is to characterize phenotypic differences among a genetically diverse population. The second step is to use gene expression microarray (or other high throughput) data from the population to construct gene co-expression networks. Co-expression analysis typically groups 20,000 genes into 20\u201330 modules containing 10\u2019s to 100\u2019s of genes, whose aggregate behavior can be represented by the module\u2019s \u201ceigengene.\u201d The third step is to correlate expression patterns with phenotype, as in GWAS, only applied to eigengenes instead of SNPs.Results and ConclusionsThe goal of the GMAS approach is to identify groups of co-regulated genes that explain complex traits from a systems perspective. From an evolutionary standpoint, we hypothesize that variability in eigengene patterns reflects the \u201cgood enough solution\u201d concept, that biological systems are sufficiently complex so that many possible combinations of the same elements (in this case eigengenes) can produce an equivalent output, i.e. a \u201cgood enough solution\u201d to accomplish normal biological functions. However, when faced with environmental stresses, some \u201cgood enough solutions\u201d adapt better than others, explaining individual variability to disease and drug susceptibility. If validated, GMAS may imply that common polygenic diseases are related as much to group interactions between normal genes, as to multiple gene mutations.R01 HL094322/HL/NHLBI NIH HHS/United StatesR21 HL110667/HL/NHLBI NIH HHS/United StatesP01 HL28481/HL/NHLBI NIH HHS/United StatesP01 HL080111/HL/NHLBI NIH HHS/United StatesHHSN268201000035C/HL/NHLBI NIH HHS/United StatesK25 HL080079/HL/NHLBI NIH HHS/United StatesP01 HL078931/HL/NHLBI NIH HHS/United StatesP01 HL028481/HL/NHLBI NIH HHS/United StatesUL1 TR000124/TR/NCATS NIH HHS/United StatesT32HL69766/HL/NHLBI NIH HHS/United StatesR01 HL070748/HL/NHLBI NIH HHS/United States1DP3 D094311/DP/NCCDPHP CDC HHS/United StatesR01 HL101228/HL/NHLBI NIH HHS/United StatesP01 HL30568/HL/NHLBI NIH HHS/United StatesR21 HL110667-01/HL/NHLBI NIH HHS/United StatesP01 HL030568/HL/NHLBI NIH HHS/United StatesT32 HL069766/HL/NHLBI NIH HHS/United StatesHHSN268201000035C/PHS HHS/United StatesR01 GM095656/GM/NIGMS NIH HHS/United States2013-08-03T00:00:00Z22859671PMC3428228vault:743

From 'omics' to complex disease: a systems biology approach to gene-environment interactions in cancer

<p>Abstract</p> <p>Background</p> <p>Cancer is a complex disease that involves a sequence of gene-environment interactions in a progressive process that cannot occur without dysfunction in multiple systems, including DNA repair, apoptotic and immune functions. Epigenetic mechanisms, responding to numerous internal and external cues in a dynamic ongoing exchange, play a key role in mediating environmental influences on gene expression and tumor development.</p> <p>Hypothesis</p> <p>The hypothesis put forth in this paper addresses the limited success of treatment outcomes in clinical oncology. It states that improvement in treatment efficacy requires a new paradigm that focuses on reversing systemic dysfunction and tailoring treatments to specific stages in the process. It requires moving from a reductionist framework of seeking to destroy aberrant cells and pathways to a transdisciplinary systems biology approach aimed at reversing multiple levels of dysfunction.</p> <p>Conclusion</p> <p>Because there are many biological pathways and multiple epigenetic influences working simultaneously in the expression of cancer phenotypes, studying individual components in isolation does not allow an adequate understanding of phenotypic expression. A systems biology approach using new modeling techniques and nonlinear mathematics is needed to investigate gene-environment interactions and improve treatment efficacy. A broader array of study designs will also be required, including prospective molecular epidemiology, immune competent animal models and <it>in vitro/in vivo </it>translational research that more accurately reflects the complex process of tumor initiation and progression.</p

The role of miRNAs in regulating the expression of flavonol pathway genes and its possible impact on the crosstalk between UV-B and flg22 signal cascades in Arabidopsis thaliana

In cell culture, we have shown that flavonol metabolite accumulation depends on expression of the Arabidopsis flavonol pathway genes (FPGs), which are upregulated by UV-B irradiation but repressed by the bacterial elicitor flg22 during MAMP Triggered Immunity (MTI). The suppression of flavonoid production during MTI is believed to allow the plant focusing its metabolism on the pathogen defense by directing phenylalanine resources from UV-B protective flavonol production towards production of phytoalexins and cell wall fortification by lignin incorporation during MTI. Here we extend our observations made initially in cell cultures by describing that this kind of signal crosstalk between UV-B and flg22 is also functional in planta. We demonstrate that such signal crosstalk is fully functional in Arabidopsis in planta. However, we observed some differences in the expression patterns of MYB transcriptions factors (TFs) as compared to data from the cell culture system. Our data suggest that in planta the TF MYB111 might play a more dominant role than the TF MYB12, which was strongly regulated in cell cultures. Thus we can present an updated working model how this crosstalk might function. We believe that this system based on seedlings of the model plant Arabidopsis thaliana constitutes a valuable platform for further dissection of the underlying molecular mechanism, e.g. by deploying gain/loss-of-function of candidate genes. Plants are confronted with various abiotic and biotic stress factors in their natural habitats, and have evolved sophisticated and multifaced mechanisms to defense themselves. In our previous works, we have demonstrated the crosstalk between flg22 and UV-B-induced signal cascades in plants, in which the expression of Arabidopsis flavonol pathway genes (FPGs) are upregulated by UV-B irradiation but simultaneously repressed by the bacterial elicitor flg22 during MAMP Triggered Immunity (PTI). Although several transcription factors proved to be involved in the crosstalk, the underlying regulatory mechanism remains largely unsolved. By deep sequencing we identified 217 miRNAs representing 204 conserved and 13 novel miRNAs involved in the crosstalk. Among them, e.g. 106 miRNAs were upregulated by flg22 and downregulated by UV-B. Furthermore, a set of specific interactions between miRNAs and their targets were confirmed showing reciprocal changes in their expression levels, simultaneously. As revealed by GO and KEGG analysis in silico, predicted miRNA target genes participate in a series of plant biological and molecular processes as well secondary metabolism pathways. Two modulations of miRNA-target interactions were obtained in the crosstalk. The first one consisting of miR158, miR165, miR166, miR167, miR168, miR172, miR391, miR393, miR447, miR824, miR828, miR846 and miR858, which were all repressed by UV-B irradiation, while upregulated by flg22, and the second one constitute miR159, miR164, miR171 and miR822, being repressed by flg22 and upregulated by UV-B. Both miRNA sets display converse regulation with the change in transcript levels of their targets. Furthermore, we demonstrated that knockdown of miR858 (Group I) in Arabidopsis goes along with increased the chalcone synthase (CHS) expression while its overexpression results in its depression. Vice versa, knockout of miR164b (Group II) depresses the CHS gene expression while its overexpression strongly elevated the CHS gene expression, providing the first genetic evidence that miRNAs identified in this study constitute an additional layer in regulating the crosstalk between flg22 and UV-B induced signaling cascades. In nature plants are often simultaneously challenged by different stress factors. The abiotic stress UV-B irradiation induces the production of UV-protective flavonols, but their accumulation is attenuated by biotic stress, e.g. by treatment with pathogen elicitors (flg22). This suppression has been shown to occur via suppression of flavonol pathway genes (FPGs) enabling the plant to direct its secondary metabolism to a more efficient pathogen defense response. Identification of two highly conserved miRNAs (miR858 and miR828) being involved in the crosstalk and their targets MYB111 and MYB75 that proved to play an important role in regulation of FPGs and the flavonoid accumulation provoked us to assume that miR858-MYB111 and miR828-MYB75 interactions play an important role in the crosstalk. Here, we demonstrate that both miR858 and miR828 are regulated by UV-B and flg22 in a UVR8 and FLS2 receptors-dependent manner and relying on the respective signaling pathways. Comparison between miR858/MYB111-promoter-GUS and their transcript levels evidences that the MYB111 is regulated not only at the transcriptional level, but also suffered from post-transcriptional modification in response to the flg22 and UV-B challenges, in which miR858 acts as a determinant regulator. Following this, we conclude that the post- transcriptional regulation mediated by plant-derived miRNAs constitutes the crosstalk between the flg22 and UV-B induced signal cascades in Arabidopsis. This allows an extension of the crosstalk model between plant responses to biotic (flg22) and abiotic (UV-B) stresses in Arabidopsis

MiRNAs as regulators of gene expression modulate development and energy metabolism of skeletal muscle

It is important to understand the molecular networks affecting biological properties of muscle in order to improve the efficiency of meat production and meat quality in domestic animals. The discovery of miRNA represents an important breakthrough in biology in recent years. MiRNA function identification has become one of the active research fields in muscle biology addressing muscle development, growth and metabolism. This thesis aims at the identification of miRNAs differentially expressed in skeletal muscle at various developmental stages and in pig breeds differing in muscularity. Moreover, links between miRNAs and mRNAs should be shown in order to address biofunctions affected by miRNAs in muscle. Finally, miRNAs impacted on muscle metabolism should be validated exemplarity by in vitro cell culture experimants. The first approach demonstrates the comprehensive miRNA expression profiles of longissimus dorsi (LD) during muscle development and growth. A comparative study on two distinct phenotypic pigs were performed using miRNA custom designed arrays. Two different key stages 63 and 91 days post-conception (dpc), and one adult stage (180 days post-natum) were analysed in German Landrace (DL) and Pietrain (Pi) breeds. Several potential candidate miRNAs are significantly up-regulated and associated with muscular developmental stages and breed types. The Affymetrix GeneChip porcine genome microarrays were also used to obtain the differential transcriptional profile of mRNA targets of the same animals. The combination of miRNA–mRNA expression data and Ingenuity Pathway Analysis established complex miRNA–dependent regulatory networks. A number of miRNA–mRNA interactions, that were associated to cellular growth and proliferation and lipid-metabolism functions, revealed insights into their role during skeletal muscle development and growth. The second approach involves in muscle growth in post mortem pig traits (crossbred [PI×(DL×DE)] population, n = 207). The experiment integrated miRNA and mRNA expression together with network analysis by using weighted gene co-expression network analysis (WGCNA). In this part, we identified the negative miRNA-mRNA co-expression networks which revealed several biological pathways underlying the difference of meat properties and muscle traits (i.e. glucose metabolic process, mitochondrial ribosome and oxidative phosphorylation). In the last approach, C2C12 in vitro model studies revealed that miRNAs are modulated in cellular ATP production and energy metabolism processes during myogenic differentiation. Correlation analyses were performed between ATP level, miRNA and mRNA microarray expression profiles during C2C12 differentiation. Among 14 significant miRNAs as representing cellular ATP regulators involved in mitochondrial energy metabolism, miR-423-3p is a novel regulator for cellular ATP/ energy metabolism via targeting the group of mitochondrial energy metabolism genes (Cox6a2, Ndufb7, and Ndufs5). In conclusion, the present study further adds a comprehensive knowledge on the systems perspective of the skeletal muscle miRNAs and their target genes regulation networks that influence on skeletal muscle starting from early muscle development to mature muscle growth.MiRNAs regulieren die Genexpression und modulieren die Entwicklung und den Energiestoffwechsel der Skelettmuskulatur Das Verständnis von molekularen Netzwerken mit Einfluss auf die biologischen Eigenschaften des Muskels ist notwendig, um die Effizienz der Fleischproduktion und die Fleischqualität in Nutztieren zu verbessern. Die Erforschung von miRNAs stellt einen entscheidenden Durchbruch in der Biologie in den letzten Jahren dar. Die Identifizierung von miRNA-Funktionen wurde seit dem eines der aufstrebenden Forschungsschwerpunkte in der Muskelbiologie mit Bezug auf Muskelentwicklung, -wachstum und -stoffwechsel. Das Ziel dieser Dissertation ist die Identifizierung von miRNAs mit differenzieller Expression in der Skelettmuskulatur im Hinblick auf verschiedene Entwicklungsstadien und Schweinerassen mit unterschiedlichem Muskelansatz. Im Weiteren soll die Verknüpfung von miRNA- und mRNA-Datensätzen helfen, durch miRNA beeinflusste Biofunktionen im Muskel zu benennen. Abschließend sollen exemplarisch einige miRNAs mit Einfluss auf den Muskelmetabolismus durch in vitro Zellkulturstudien validiert werden. Der erste Forschungsansatz lieferte umfassende miRNA-Expressionsprofile des longissimus dorsi (LD) während der Muskelentwicklung und des Wachstums. Dazu wurden Schweine mit unterschiedlicher phänotypischer Ausprägung unter der Verwendung von spezifisch gefertigten miRNA-Arrays vergleichend analysiert. Tiere der Deutschen Landrasse (DL) und der Rasse Pietrain (Pi) wurden zu zwei wesentlichen pränatalen Entwicklungszeitpunkten (am 63 und 91 Tag nach Empfängnis) sowie im adulten Stadium (180 Tage nach Geburt) untersucht. Für zahlreiche potentielle Kandidaten-miRNAs konnte gezeigt werden, dass diese signifikant hochreguliert sind und Assoziationen zu muskulären Entwicklungsstadien und der Rasse aufzeigten. Zusätzlich wurden porcine Genommikroarrays (Affymetrix GeneChip) verwendet um Profile der differentiell exprimierten mRNA-targets im gleichen Tier zu untersuchen. Durch die Kombination von miRNA- und mRNA-Expressionsdaten gekoppelt mit Ergebnissen aus der Analyse von regulierten Signalwegen (Ingenuity pathway analysis) konnte ein Komplex aus miRNA-abhängigen regulatorischen Netzwerken etabliert werden. Zahlreiche miRNA-mRNA-Interaktionen im Zusammenhang mit Funktionen des zellulären Wachstums, der Proliferation und des Fettstoffwechsels, ermöglichten Einblicke in die Funktion dieser Wechselwirkungen während der Entwicklung und des Wachstums der Skelettmuskulatur. Der zweite Forschungsansatz berücksichtigt das Muskelwachstum in relevanten post mortem Merkmalen (Kreuzungsrasse [Pi x (DLxDE), n=207). Für diesen Ansatz wurden die Expressionsdaten der miRNA- und mRNA-Analysen in einem Ko-Expressionsnetzwerk integriert. Dabei wurden die Wechselwirkungen zwischen verschiedenen Komponenten berücksichtigt und gewichtet. Negative miRNA-mRNA-Ko-Expressionsnetzwerke konnten identifiziert werden. Diese deuten auf biologisch relevante Signalwegen hin, welche mit unterschiedlichen Ausprägungen der Fleischeigenschaften und Merkmalen der Muskulatur in Zusammenhang stehen (z.B. Prozesse des Glucosemetabolismus, mitochondriale Ribosomen und oxidative Phosphorylierung). Im abschließenden Forschungsansatz konnte durch Analysen des C2C12-Muskelzellmodells gezeigt werden, dass miRNAs im Zusammenhang mit der zellulären ATP-Produktion und mit Prozessen des Energiemetabolismus im Rahmen der myogenen Differenzierung reguliert werden. Dazu wurden zum Zeitpunkt der C2C12-Zelldifferenzierung ermittelte ATP-Gehalte und miRNA- und mRNA-Mikroarray-Expressionsprofile miteinander verknüpft. Unter den 14 miRNAs, die als zelluläre ATP-Regulatoren am mitochondrialen Energiemetabolismus beteiligt sind, konnte miR-423-3p, durch den Einfluss auf Gene aus der Gruppe des mitochondrialen Energiemetabolismus (Cox6a2, Ndufb7 und Ndufs5), als neuer Regulator für zelluläres ATP bzw. den Energiemetabolismus bestätigt werden. Zusammenfassend liefern die vorliegenden Studien wesentliche Erkenntnisse zu systemischen Funktionen der miRNAs in der Skelettmuskulatur und verdeutlichen ihren Einfluss auf Gennetzwerke, welche die Prozesse von der frühen Muskelentwicklung bis hin zum Muskelwachstum beeinflussen