MONDAY, MARCH 22
8:00-8:30 am Registration for Pre-Conference Short Course
8:30 am -12:00 pm Pre-Conference Short Course*
(*Separate Registration Required)
8:30-8:35 am Chairperson’s Opening Remarks
8:35-9:00 Direct, Electronic microRNA Profiling on a Multiplexed Chip
Shana O. Kelley, Ph.D., Professor, Biochemistry, University of Toronto
We have developed an electronic chip featuring nanostructured microelectrodes (NMEs) that enables the analysis of microRNA expression profiles in 30 minutes in small RNA samples without enzymatic amplification or sequence labeling. The multiplexed chip detects the hybridization of microRNA targets to NME surfaces and provides large electrocatalytic gain through the use of an ultrasensitive redox reporter system. Using this device, we identified several microRNA sequences that are overexpressed in cancer cells. This technology has the potential to enable the high-throughput identification of microRNAs for biomarker identification or cancer diagnosis.
9:00-9:25 The Regulation of microRNA Biogenesis in Cancer and Development
J. Michael Thomson, Ph.D., Assistant Professor, Department of Cancer Biology, Vanderbilt-Ingram Cancer Center
microRNA expression is highly tissue-specific and is tightly regulated, both spatially and temporally, in normal development. Additionally, microRNA dysregulation has proven to play a pivotal role in the initiation and progression of a wide variety of human cancers. It was long assumed that the strict control of microRNA expression was attributable to transcriptional regulation, but it’s become increasingly clear that, at least for a subset of microRNAs, expression is regulated at a post-transcriptional level via differential processing. Our lab has developed novel tools to address the post-transcriptional regulation of microRNA biogenesis.
9:25-9:50 Integrating Micro, Messenger and Non-Coding RNA Expression Analysis via a Single Array Profiling Platform
Xiaolian Gao, Ph.D., Professor, Biology and Biochemistry, Chemistry, and Biomedical Engineering, University of Houston
The importance of microRNA profiling in disease pathway study and diagnostics has been well recognized. A basic biological function of microRNAs (miRNA) is gene regulation through interacting with messenger RNAs (mRNA). In order to understand the regulatory networks it is extremely important to have profiling information on both types of RNAs. However, up to now the profile measurements of the two RNAs have been done separately, often on different platforms. This makes it difficult to reach a unified assessment of the two RNAs. For example, the relative copy numbers of the two RNAs are critical for studying their interaction kinetics but the normalization links between two different assays are often missing. Here we present simultaneous profiling of miRNA, mRNA and other non-coding RNAs from a single sample on an advanced microfluidic microchip platform. A ligation-based assay is used to ensure that labeling reaction works for all RNAs with or without poly-A tails; each RNA transcript has one labeling tag so that signal intensity is proportional to transcript copy numbers; and the reaction does not involve random priming so that transcripts of different lengths have equal chance for the reaction. Clustered dyes are used in signal amplification for enhancing detection sensitivity. The availability of this unified profiling method will significantly enhance our ability to identify regulatory networks, expand the scope of information for disease pathways studies, and provide multiple-category biomarkers for disease diagnosis.
9:50-10:15 A High-Content Assay to Screen for Modulators of the miRNA Machinery
David Shum, Ph.D., Assay Development Specialist, HTS Core Facility, Memorial Sloan Kettering Cancer Center
microRNAs are small non-coding regulatory RNAs reducing stability and/or translation of fully or partially sequence-complementary target mRNAs in plants and animals. Their upregulation has been reported in many malignancies and proposed to regulate several tumor suppressor pathways; the understanding of their biogenesis may offer novel therapeutic approaches and intervention. There are no well-documented modulators to help us study the pathway and unravel its biological significance; for this purpose, we set out to develop a high-content assay using a stably transfected HeLa S3 cells expressing EGFP under miR-21 regulation as the reporter gene carries a sequence with perfect complementarity to miR-21 in its 3‘ UTR region, thus decrease in miR-21 levels would induce increase in the expression and maturation of the EGFP reporter which can easily be imaged using automated microscopy. We will present and discuss our assay development and validation against both a pilot chemical library and a custom siRNA library targeting known genes of the miRNA pathway together with few essential housekeeping genes to assess transfection efficiencies. Our high-content assay and its dual adaptation would allow screening and discovery of novel modulators of the miRNA pathway and a better understanding of the regulatory components of its maturation machinery.
10:15-10:45 Networking Coffee Break
10:45-11:10 Nuclear miRNAs and Cytoplasmic miRNAs
Clark Jeffries, Ph.D., Research Professor, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill
We report discovery of two classes of miRNAs relative to one cell system, namely, miRNAs that preferentially accumulate in the nucleus or in the cytoplasm of human embryonic neural progenitor cells. While it is known that many miRNAs appear in both nucleus and cytoplasm, we used TaqMan® technology to yield accurate measurements showing that certain miRNAs have preferred localization. Remarkably, if one miRNA is localized, then other miRNAs that have both similar 5’ end patterns and similar 3’ end patterns tend also to be localized in the same way. If nuclear import of mature miRNAs is part of a feedback mechanism that regulates miRNA levels, then importation dysregulation might correlate with disease.
11:10-11:35 microRNAs and Breast Cancer Metastasis: The Power of Pleiotropy
Scott Valastyan, Ph.D., Laboratory of Robert A. Weinberg, Massachusetts Institute of Technology and Whitehead Institute for Biomedical Research
microRNAs are well-suited to regulate tumor metastasis due to their capacity to concomitantly inhibit numerous target genes, thereby potentially enabling their simultaneous intervention at multiple distinct steps of the metastatic process. We have identified a microRNA exemplifying these attributes, miR-31, whose expression levels correlated inversely with metastatic recurrence in human breast cancer patients. Ectopic expression of miR-31 in otherwise aggressive breast tumor cells impeded metastasis. We deployed a novel microRNA sponge strategy to stably inhibit miR-31 in vivo; this allowed otherwise non-aggressive breast cancer cells to metastasize. These effects were achieved via pleiotropic modulation of a cohort of clinically relevant motility- and metastasis-promoting genes, which were overrepresented among the >200 mRNAs computationally predicted to be direct downstream targets of miR-31. In fact, we discovered that miR-31-evoked concurrent regulation of three such effectors – integrin 5, radixin, and RhoA – which was sufficient to explain the full spectrum of this microRNA’s impacts on metastasis. Taken together, these findings provide mechanistic insights into tumor metastasis and have implications concerning the importance of pleiotropy for the biological actions of microRNAs.
11:35 am-12:00 pm Development and Validation of a miRNA-Based Laboratory Developed Test (LDT) for Pancreatic Cancer
Anna Szafranska-Schwarzbach, Ph.D., CLIA Laboratory Supervisor, Pharmacogenomics Services, Asuragen, Inc.
We have previously reported a laboratory-developed test (LDT) based on differential expression of miR-196a and miR-217 that can distinguish pancreatic ductal adenocarcinoma (PDAC) from chronic pancreatitis with a sensitivity and specificity of ~95%. This test performs well on specimens with either large (≥60%) tumor area or those that can be enriched to this tumor content. There is additional clinical interest in identifying small pancreatic malignancies in a background of pancreatitis, particularly as a step toward early detection in tissues. We will discuss the LDT validation and the identification of a new miRNA classifier that allows discrimination of PDAC in specimens with low suspected tumor content.
12:00 Close of Pre-Conference Short Course