Identification of Normal and Neoplastic Human Mature B Cells “miRome”.

The recent discovery of microRNAs (miRs) has added a new dimension to the mechanisms that regulate gene expression in normal cell development. Initial evidence also suggests that structural or functional alterations of miRs are associated with tumorigenesis. Nonetheless, the full set of genomic miRs is not known for any eukaryotic species including humans, since presently known miRs have been identified by virtue of their expression in only a few cell types. Analogous to other transcriptional units, it is conceivable that a significant part of the entire miR genomic pool may display tissue-specific expression. In order to understand the role of miRs in mature B cell function and lymphomagenesis, we planned the isolation of the full set of miRs (miRome) expressed in germinal center (GC) B cells and in GC-derived B cell lymphomas. Toward this end, we constructed libraries representative of all miRs expressed in normal GC B cells isolated from human tonsils and in a Burkitt lymphoma cell line (Ramos). Short RNAs were gel purified and linked to adaptor oligonucleotides, reverse transcribed, PCR amplified, cloned into an expression vector, and subjected to sequencing. Candidate miRs have been aligned to the human genome to retrieve their potential precursor sequences, whose folding characteristics have been analyzed in order to identify bona fide precursors. The cloned and structurally validated miR precursors have been matched to the public miR database to detect previously identified miR sequences. Initial results show that 453 miRs cloned from GC B cells correspond to 99 precursors (59 previously known and 40 newly discovered), while 233 miRs cloned from Ramos cell line identified 42 known and 46 new precursors. Overall, this initial analysis led to the discovery of 76 not previously reported miR precursors. Since less than a third of the available clones have been analyzed, it is expected that a significantly larger number of novel miRs will be identified by our method. A comprehensive analysis of miR expression profiles of normal versus neoplastic GC B cells will be obtained using a dedicated miR chip. Interestingly, initial results indicate that a set of miRs is expressed in normal GC cells, but not in Ramos and other GC-derived Burkitt lymphoma cell lines, suggesting that structural and/or functional alterations of miRs occur during lymphomagenesis.