Genome-wide analysis of microRNAs and their targets in CLL

MicroRNAs (miRs) are small, non-coding RNAs, which act as regulators of the expression of protein-coding genes and are thereby involved in cellular and tissue development, as well as in processes like cell proliferation and apoptosis. By this means, miRs can also act as tumour suppressor or oncogenes. In 2002, the first association of miRs and cancer was described for CLL. Since then, the relevance of miRs during the development and progression of a variety of cancer entities was discovered. Via microarray-based techniques and quantitative RT-PCR we detected the differential expression of known and novel miRs in primary CLL cells compared to normal B cells. Functional investigations of deregulated miRs revealed that miR-130a controls autophagy and thereby cell death in CLL cells. We further identified several so far unknown target proteins of miR-130a, and showed that one of these, Dicer1, is of relevance in this process (see Kovaleva et al., 2012).
To understand the function of miRs in tumorigenesis, it is essential to know their targets. Current algorithms to predict target genes based on sequence homologies are imperfect, since only a part of the miR-targets can be identified and in many cases the prediction cannot be verified biochemically. Therefore, we aimed at a genome-wide analysis of miR-targets in primary CLL cells and established a novel method that is based on immunoprecipitation of RNA-interacting protein (in our case AGO2, a main component of the miR processing complex) followed by next generation sequencing of the co-precipitated RNA fraction (RIP-Seq). This technique allows for the identification of the entire targetome of cells, as well as a genome-wide identification of targets of single miRs after their ectopic expression. We have used this technique to characterize the targetome of miR-155, a miR with oncogenic potential that induced enhanced proliferation in our cell line model. In addition, we have obtained and compared the miRnome and targetome data of two non-Hodgkin B cell lymphoma lines (MEC-1 and JEKO-1) and thereby identified candidate genes which are currently analysed concerning their function in B cell malignancies. Integrative analysis of these data confirmed the relevance of miR-155 in regulating cell proliferation and suggested some novel target genes that might be involved. In addition, a disproportional binding of specific miRs to AGO2 was detected and is recurrent in all cell types analysed so far, which suggests a novel control mechanism of miR activity (see Meier et al., 2013).
Currently, we are using RIP-Seq to identify the miRnome and targetome of primary CLL and MCL cells and to integrate these data with those of the cell lines. The final goal of these studies is the identification of disease-relevant miRNAs and their targets in CLL and MCL and a subsequent evaluation of their potential for novel therapeutic approaches.

RIP-Seq identifies novel miR-155 targets
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Immunoprecipitation of AGO2 protein was used to isolate associated miRNA and target transcripts, which were subsequently identified by miRNA TagMan arrays and mRNA sequencing, respectively. By this means we detected 100 transcripts that were enriched in the IP fraction upon ectopic miR-155 expression in HEK-293T cells. Several of these genes are potential candidates to explain the enhanced proliferation observed in these cells. In addition, a recurrent disproportional loading of specific miRNAs in AGO2 complexes was observed in HEK cells as well as the two B cell lines JEKO-1 and MEC-1 (Meier et al, 2013).

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