TUESDAY, MARCH 24
7:30-8:00 Morning Coffee
microRNA in Development: Stem Cells and Beyond
8:00-8:05 Chairperson’s Opening Remarks
Richard Gregory, Ph.D., Assistant Professor, Department of Biological Chemistry and Molecular Pharmacology, Harvard Stem Cell Institute, The Stem Cell Program at Children’s Hospital Boston, Harvard Medical School
8:05-8:30 Regulation of miRNA Biogenesis in Embryonic Stem Cells and Cancer
Richard Gregory, Ph.D., Assistant Professor, Department of Biological Chemistry and Molecular Pharmacology, Harvard Stem Cell Institute, The Stem Cell Program at Children’s Hospital Boston, Harvard Medical School
Posttranscriptional control of miRNA biogenesis has been observed in embryonic stem (ES) cells, embryonal carcinoma (EC) cells, and primary tumors. These data support the notion that unidentified mechanisms exist in stem cells and certain cancers to prevent miRNA-mediated cell differentiation. We recently identified the developmentally regulated RNA-binding protein Lin28 as a selective inhibitor of the maturation of let-7 family miRNAs in embryonic cells. We seek to understand the mechanisms by which Lin28 controls miRNA biogenesis, as well as to identify novel miRNA regulatory pathways.
8:30-8:55 microRNA Function in Human Embryonic Stem Cells
Hannele Ruohola-Baker, Ph.D., Professor, Biochemistry, University of Washington
We show that knockdown (KD) of Dicer or Drosha gene products dramatically attenuates cell division in human embryonic stem cells (hESCs). Using microarray and QPCR analysis we identified a hESC enriched microRNA (miRNA) group. Work is underway to test which of these miRNAs can rescue the Dicer KD cell division phenotype. Interestingly, we have already identified two miRNAs that can partially rescue the cell division defects observed in hESC dicer KD lines.
8:55-9:20 Beyond the 3’UTR: Expanded Roles for Human and Mouse microRNAs
Isidore Rigoutsos, Ph.D., Manager, Bioinformatics & Pattern Discovery Group, Computational Biology Center, Deep Computing Institute, IBM Thomas J. Watson Research Center
According to the standard model of microRNA activity in animals, microRNAs operate by acting on the 3’UTR of the targeted transcript. Moreover, according to the model, sequence conservation across genomes is an important characteristic of a microRNA target. In this presentation, I will describe and discuss recent work showing that in humans and mice microRNAs can also target locations of a mRNA transcript that are outside the 3’UTR; notably, these targeted locations are not conserved across genomes. We conjecture that microRNA activity outside the 3’UTR and at non-conserved locations may in fact be more frequent than the standard model anticipates.
9:20-9:45 microRNA in Mesenchymal and Embryonic Stem Cells
Mark Landers, Ph.D., Scientist, Research & Development, Advanced Sequencing Applications, Invitrogen
We will present results on expression of miRNA in embryonic stem cells and mesenchymal stem cells using array and sequencing technology. We will discuss results on the role of various miRNA in regulating growth factor and receptor expression. We will present results on the change in miRNA expression as MSC differentiate into adipocytes, chondrocytes and osteocytes.
9:45-10:30 Coffee Break with Exhibit and Poster Viewing
| Technology Showcase |
Sponsored by
.JPG "febit new") 10:30-10:45 MPEA a miRNA Profiling Method Using On-Chip Signal Amplification; Ideal for Small Sample Amounts
Michelle Lyles, Ph.D., Vice President, Marketing & Sales, Febit Inc.
Small noncoding RNAs (sncRNAs) have moved from a curiosity to a key albeit poorly understood component of gene regulation. Next Generation Sequencing has been at the forefront in the discovery of many such noncoding molecules, yet flexible tools translating this sequence information into affordable high throughput assays are lacking. Here we describe a microfluidic primer extension assay (MPEA) for the detection of sncRNAs employing highly flexible microfluidic microarrays. MPEA is unique in its integration of conventional hybridization with on-chip enzymatic elongation (primer extension). sncRNAs can be detected in little as 20 ng of total RNA. Importantly, MPEA does not require labeling prior to hybridization and – due to its high sensitivity –amplification is not required. Thus, no bias is introduced by such processes. Here, we present detection of miRNA, and the flexibility of the technology platform enables analysis of any sncRNA, such as piRNAs.
Sponsored by
 10:45-11:00 miRNA Profile as a Prognostic Marker for Chemopreventive Resistance
Jennie L. Williams, Ph.D.,Cancer Prevention Laboratory,State University of New York at Stony Brook
We have demonstrated that promising colon cancer chemopreventive agents, derivatives of Non-steroidal anti-inflammatory drugs (NSAIDs), inhibits colon cancer cell growth by inhibiting cell proliferation and enhancing cell killing. However, very little is known about the molecular targets in the cancer cell that are responsible for this effect. microRNAs (miRNAs), small (17- 22 nt) regulatory RNAs, are known to control gene expression and translational repression or degradation, via specific sites at the 3’-UTR of its target mRNAs. The deletion or mutation of one particular miRNA could potentially lead to the progression of disease; specifically, cancer. Therefore, using total RNA isolated from tumors of xenographic mouse and colorectal cancer cells treated with NSAID derivatives, miRNA distribution patterns were determined and analyzed by Marligen’s multiplex bead arrays. Protein lysates of the tumors and tissue culture cells were also examined for levels of transcriptional activating factors (i.e., NF-B and p53). Our hypothesis, based on preliminary results, is that difference in miRNA expression/regulation may account, at least in part, for the differential response to chemoprevention. The clinical response to chemopreventive agents often differs between populations of individuals. Since chemopreventive agents exert their effect through a molecular target, this difference would suggest irregularity at the genetic level. Consequently, the generation and evaluation of chemopreventive agents that address the issue of chemo-resistance is essential. Thus, a correlation of altered level of miRNA and the expression and/or activation of its target genes may ascertain its association with poor clinical outcome.
Sponsored by
 11:00-11:15 The role of miRNA’s in the Differentiation of Human Mesenchymal Stem Cells to Osteoblasts
Kirsteen. H. Maclean, Field Application Scientist, Dharmacon, Thermo Fisher Scientific
MicroRNAs (miRNAs) operate as key regulators of gene expression by repressing mRNA translation or targeting transcripts for degradation. Recent reports on the role of miRNAs in development and differentiation suggest novel opportunities to employ synthetic miRNA mimics and inhibitors as molecular tools in a variety of applications. These applications include miRNA expression profiling for prognostic or diagnostic indicators of normal or disease states or screening with synthetic miRNA mimics and inhibitors to elucidate key regulators of specific biological pathways. In this talk we’ll review examples of a miRNA-based inhibitor screen to highlight the utility of these tools in basic biology and drug discovery platforms to identitify miRNAs involved in the differentiation of human bone marrow mesenchymal stem cells into osteoblasts. Further approaches such as the use of miRNA mimics and profiling strategies highlight the importance of hit stratification and validation.
Sponsored by
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11:15-11:45 Leading the Way in MicroRNA: Discovery, Validation and Functional Analysis
David Ruff, Ph.D., Applied Biosystems
MicroRNA (miRNA) has been identified as an important regulator of gene expression. These small noncoding RNA molecules modulate gene expression through post-transcriptional interactions with specific mRNA targets. A total of 695 miRNA genes have been identified to date in humans. Many researchers believe that at least few hundreds of additional novel miRNAs are yet to be discovered but conventional cloning and Sanger sequencing method may not meet this increasingly difficult task. We have developed road maps and tool kits to approach the discovery, validation and functional analysis of miRNA. Beginning with sample preparation strategies that enable the construction of tagged highly complex small RNA libraries suitable for the SOLiDTM platform, these workflows greatly facilitate the discovery of novel small RNA molecules. Employment of the high-throughput and sensitive gold standard TaqMan® MicroRNA Assays provide an expression profile for hundreds of miRNAs from sub-nanogram quantities of total RNA in as little as five hours. Application examples using these tools for miRNA profiling reveals unique signatures for Acute Myeloid Leukemia and ES single cells. To further elucidate miRNA function, combinations of miRNA mimics and inhibitors help identify gain- and loss-of function phenotypes. When combined with TaqMan® Gene Expression Assays and protein expression tools, coordinate investigation of mRNA, miRNA and protein levels give a more global understanding of cellular biology. Our innovative systems enable researchers to address fundamental questions about miRNA science and their key role in disease processes.
11:45-12:00 Sponsored Presentation
(Opportunity available)
Contact Jon Stroup, Manager, Business Development, at jstroup@healthtech.com or 781-972-5483
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12:00-1:30 Lunch on your own
microRNA in Health and Disease
1:30-1:35 Chairperson’s Opening Remarks
Neil Smalheiser, M.D., Ph.D., Associate Professor, Psychiatry, University of Illinois at Chicago
1:35-2:00 Expression of microRNAs and their Precursors in Synaptic Fractions of Adult Mouse Forebrain
Neil Smalheiser, M.D., Ph.D., Associate Professor, Psychiatry, University of Illinois at Chicago
This presentation shows that certain microRNAs are enriched near synapses, and that pre-miRs are similarly localized in association with post-synaptic densities. Dicer and Ago proteins are also enriched in this location, and dicer is liberated/activated by synaptic activity. This suggests that pre-miRs are transported to dendrites and that miRs are formed (at least in part) locally in dendritic spines in an activity dependent manner.
2:00-2:25 Regulation of the Immune System by microRNA-155
Elena Vigorito, Ph.D., Group Leader, The Babraham Institute, UK
MicroRNAs (miRNAs) represent a new class of regulatory genes important for a variety of cellular processes including cell growth, differentiation and lineage commitment. In the immune system, miRNAs affect myeloid and lymphoid differentiation and function. A particular miRNA, miR-155, is rapidly induced in lymphoid and myeloid cells upon activation. Moreover, it is highly expressed in hematological malignancies and it causes myeloid proliferation and B cell expansion when over-expressed in mice. Unraveling how the expression of miRNAs is regulated, which cellular function they influence and identification of their molecular targets under normal conditions is therefore essential to understand their role in cancer. To define the role of miR-155 in vivo, we have analyzed the phenotype of mice in which miR-155 has been ablated. In this presentation, I will summarize what we have learned on the regulation of the immune system by miR-155.
2:25-2:50 microRNAs in an Autoimmune Disease Animal Model
Marianthi Kiriakidou, M.D., Assistant Professor, Medicine, University of Pennsylvania School of Medicine
Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease characterized by autoreactivity of B and T cells, production of autoantibodies and deposition of immune complexes, resulting in multiple organ damage. Although the molecular and cellular pathogenesis of SLE is poorly understood, there is growing evidence that B and T lymphocytes play a key role. The vertebrate adaptive immune system has evolved a number of cellular ‘checkpoints’ along the lymphocyte maturation pathway designed to eliminate self-reactive B and T cell clones. Failure to establish self-tolerant B or T cells at any of these checkpoints results in autoreactive clones. microRNAs are a novel class of small, non-coding RNAs that regulate gene expression by blocking translation and/or promoting degradation of cognate target mRNAs. microRNAs have been shown to regulate numerous cellular and developmental processes. Their role in the regulation of the immune system, although a subject of intense investigation, is largely unknown. To address the role of microRNAs in the pathogenesis of SLE, we profiled the expression of microRNAs in FACS purified B and T lymphocytes from the lupus-prone, tri-congenic mouse model, B6.Sle123. Our findings show that among the microRNAs that are differentially expressed in B6.Sle123 derived B and T cells, several microRNAs with key roles in the development and function of lymphocytes are included. We are currently investigating whether these and other differentially expressed microRNAs target components of the B and T cell maturation checkpoints contributing to the proliferation of autoimmunity.
2:50-3:45 Refreshment Break with Exhibit and Poster Viewing
3:45-4:10 microRNAs Induced During Adipogenesis that Accelerate Fat Cell Development are Downregulated in Obesity
Harvey F. Lodish, Ph.D., Member, Whitehead Institute for Biomedical Research; Professor, Biology & Bioengineering, Massachusetts Institute of Technology
We investigated the regulation and involvement of microRNAs (miRNAs) in fat cell development and obesity. We profiled and validated the expression of more than 370 miRNAs during adipogenesis of preadipocyte 3T3-L1 cells, mature normal primary epididymal adipocytes, purified preadipocytes, and adipocytes from leptin deficient ob/ob and diet-induced obese mice. The same miRNAs were differentially regulated during adipogenesis both in culture and in vivo. Importantly, miRNAs that were induced during adipogenesis were downregulated in adipocytes from both types of obese mice, and vice versa. These changes are likely associated with the chronic inflammatory environment as they were mimicked by TNFa treatment of differentiated adipocytes. Ectopic expression of two regulated miRNAs in preadipocytes accelerated adipogenesis, were measured both by the upregulation of many adipogenesis markers and by an increase in triglyceride accumulation at an early stage of adipogenesis. An understanding of the role of miRNAs in adipose biology may lead to novel RNA-based therapies that complement current anti-obesity treatments.
4:10-4:35 The Myriad Roles of microRNAs in Heart Disease
Eva van Rooij, Ph.D., Director of Pre-Clinical Research, miRagen Therapeutics, Inc.
The heart responds to diverse forms of stress by hypertrophic growth and reprogramming of cardiac gene expression, which culminate in a loss of pump function, arrhythmias, and sudden death. We have identified a signature pattern of microRNAs that are dysregulated during pathological cardiac remodeling and heart failure in both mouse and human models of heart disease. Gain- and loss-of-function studies in mice have revealed profound and unexpected functions for these microRNAs in the heart, including the control of myocyte growth and identity, contractility, energy metabolism, and stress responsiveness, providing glimpses of new regulatory mechanisms and potential therapeutic targets for heart disease. The prominent functions of microRNAs provide unique therapeutic opportunities for manipulating microRNA biology in the setting of cardiovascular disease.
4:35-5:00 microRNAs as Biomarkers of Alzheimer’s Disease
John P. Cogswell, Ph.D., Genetics Consultant, GlaxoSmithKline
MicroRNAs have essential roles in brain development and neuronal specification but their roles in neurodegenerative diseases such as Alzheimer’s disease (AD) are unknown. Using a sensitive qRT-PCR platform we identified regional and stage-specific deregulation of miRNA expression in AD patient brains. We used experimental validation in addition to literature to reveal how the deregulated brain miRNAs are biomarkers for known and novel pathways in AD pathogenesis. We additionally recovered miRNAs from cerebrospinal fluid and discovered AD-specific miRNA changes consistent with their role as potential biomarkers of disease.
5:00 Close of Day