Identification of miRNA-target proteins in mantle cell lymphoma (MCL)
Dr. Armin Pscherer
Fig.: Transgenesis-System for miRNA-Expression: RMCE (Recombinase-Mediated Cassette Exchange) Strategy. The selection cassette coded by the targeting vector is integrated randomly into the genome and selected by neomycin resistance to monclonality. Both expression vectors, Pol II- and Pol III-driven versions, were modified with the two respective lox-sites, obtaining the pCMXdL and pSUPERdL-versions („dL“ for „double Lox“). By cotransfection of a Cre-expression plasmid (pCMXhCre) and either a pCMXdL-PolII or a pSUPERdL-PolIII, the single-copy transgen between the loxP511- and loxP-sites is exchanged, selectable by an HSV-Tk counterselection with ganciclovir (GCV).
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The goal of this multidisciplinary project is the establishment, optimization and validation of the proteomics-approaches SILAC (stable isotope labeling with amino acids in cell culture) and ICPL (isotope coded protein label) as screening tools for target proteins, which are translational repressed by distinct miRNAs (Fig.). The specific haematopoietic expression of distinct miRNAs in addition to their genomic aberration status in MCL points to a potential role of these miRNAs during normal haematopoiesis and leukaemogenesis. We are able to test the impact of distinct miRNAs and their target proteins on the deregulated cell phenotype of MCL-cells by expressing miRNA-candidates via a stable, well-controllable transgenesis-system (Fig.). In order to understand the biological function of miRNAs and their impact on carcinogenesis, it is essential to identify the target proteins regulated by miRNAs.
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The mass-spectrometry based quantification approaches SILAC and ICPL are used for the analysis of disease-associated miRNAs, which are identified in MCLs as new or cell-type specific. In-silico bioinformatics based prediction algorithms are currently applied to narrow down potential miRNA-target genes. In contrast an experimentally based approach to identify miRNA target proteins could not be established so far. Outstanding candidates of MCL-specific miRNAs will be ectopically expressed in MCL-cells to check for their potential to posttranscriptionally regulate a target protein. By this new approach, mass spectrometry based analysis and quantification of miRNA target proteins, we will contribute to the understanding of the pathomechanism controlling this heterogenous tumor entity.
Fig.: Scheme showing the SILAC method. Two seperate cell clones (one overexpressing a distinct miRNA, compared to a control clone) are cultured in media containing light (12C6-) and heavy (13C6-) lysine, respectively. Following isolation of microsomal proteins, equal amounts of protein from each sample are combined, creating a single sample that is then separated by SDS-PAGE. The entire gel lane is divided into 13 regions, and each section is processed for mass spectrometry. Quantitative data are determined from the ratio of peptide intensities produced from the light and heavy peptides in the MS spectra. From: Everley, P. A., Krijgsveld, J., Zetter, B. R. and Gygi, S. P. Quantitative Cancer Proteomics: Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) as a Tool for Prostate Cancer Research. (2004). Molecular and Cellular Proteomics. 3, 729-735.
Cooperation Partners;
Prof. Dr. Hartmut Döhner, PD Dr. Stephan Stilgenbauer, Division Internal Medicine III, University of Ulm
group Protein Analysis Facility of Dr. Martina Schnölzer, DKFZ