title: Elucidation of regulatory micro-RNA/ messenger-RNA networks in B-cell chronic lymphocytic leukemia and mantle cell lymphoma
creator: Meier, Jan
subject: ddc-500
subject: 500 Natural sciences and mathematics
description: B-cell chronic lymphocytic leukemia (CLL) is the most common type of leukemia in the western world. CLL is not curable with currently available therapies. Another neoplastic entity that shares common genotypic and phenotypic characteristics with CLL is mantle cell lymphoma (MCL). Like CLL, MCL is an incurable B-cell neoplasm comprising approximately 6% of all Non-Hodgkin lymphomas (NHL).  MicroRNAs (miRNAs) are small single stranded RNA molecules with a length of ~21 nucleotides, which regulate the stability and translation of protein coding messenger RNAs (mRNAs). It is estimated that 30% of all protein coding genes of an organism are regulated by miRNAs. Within recent years, evidence arose that the deregulated expression of miRNAs plays a pivotal role in cancer.  MicroRNA-155 and the miR-17-92 cluster are aberrantly expressed in various cancers including B-cell lymphomas. The present work aimed to identify transcripts regulated by these aberrantly expressed miRNAs. To this end, current standard techniques like quantitative real-time reverse transcription PCR, luciferase sensor assays and Western blot analyses were performed. As the results of these studies revealed just minor effects, an alternative screening method was established, which is based on the immunoprecipitation (IP) of RNA induced silencing complexes (RISC) with subsequent sequencing of precipitated mRNAs (RIP-Seq).  As a model system for RIP-Seq, HEK293T cells with stable, ectopic expression of miR-155 were generated. In comparison to the control cell line, these cells showed enhanced cell proliferation. RIP-Seq experiments using these cells revealed an enrichment of 67 mRNAs predicted as miR-155 targets, including related to cell proliferation. Some of these, like the transcription factor CEBPB, are well established miR-155 target genes. Others like the RNA destabilizing protein ZFP36 are frequently reduced in human cancers. Expression profiling further revealed significant changes in the transcriptome and miRNome of these cells, presumably as a secondary consequence of ectopic miR-155 expression. For instance, the cell cycle gene CCND1 was severely up-regulated after miR-155 overexpression. Furthermore, the cancer associated ETS transcription factor family PEA3 was highly up regulated as well. Using RIP-Seq, the targetomes of two B-cell lines representing CLL and MCL were identified. These targetomes comprise more than 1,000 target genes each, with more than 600 genes shared by both cell lines. The putative tumor suppressor gene BTG2 was one of the most prominent miRNA target genes identified by RIP-Seq in both cell lines. BTG2 was predicted to be simultaneously regulated by more than 15 of the 30 most highly expressed miRNAs within these cell lines.  MicroRNA profiling of IP and corresponding total lysate fractions generated by RIP-Seq, uncovered a recurrent disproportional presence of several miRNAs, including miR-155 and members of the miR-17-92 cluster, within the AGO2 IPs. However, the underlying mechanisms, explaining the bias in miRNA binding to AGO2 remain elusive so far. 
date: 2012
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/13610/1/Doctoral_Thesis_Jan_Meier.pdf
identifier: DOI:10.11588/heidok.00013610
identifier: urn:nbn:de:bsz:16-heidok-136109
identifier:   Meier, Jan  (2012) Elucidation of regulatory micro-RNA/ messenger-RNA networks in B-cell chronic lymphocytic leukemia and mantle cell lymphoma.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/13610/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng