Investigating Mature B-Cell Lymphomas by MicroRNA Profiling.

Abstract

In order to understand the role of microRNA (miRNAs) in mature B cell function and lymphomagenesis, we analyzed the miRNA expression profile of B cells at different stages of differentiation by cloning of short RNA and by array-based miRNA expression studies. We generated libraries representative of miRNAs expressed in normal germinal center (GC), naïve and memory 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 miRNAs 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 miRNA precursors have been matched to the public miRNA database (miRBase v10.0) to detect previously identified miRNA sequences. The results show that 201 (109 previously known and 92 newly discovered) mature miRNAs were cloned from normal B cells and Ramos cell line. Overall, this analysis led to the discovery of 92 not previously reported miRNAs and allowed us to identify the complete miRNA expression profile of different stages of B cell development. The cloning data show a good degree of concordance with the array-based analysis leading to the identification of a distinct miRNA expression profile for each normal B cell population. Naïve and memory B cells appear to share a large fraction of their miRNA profile while centroblasts display a clearly unique pattern of miRNA expression. We performed a comprehensive analysis of miRNA expression profiles of GC-derived lymphomas (Burkitt lymphoma, diffuse large B cell lymphoma, follicular lymphoma) using the same microarray platform including approximately 500 miRNA sequences. Each tumor type shows a distinct profile that separates them from their normal counterpart. Interestingly, initial results indicate that a set of miRNAs is expressed in normal GC cells, but not in lymphoma samples and cell lines, suggesting that structural and/or functional alterations of miRNAs occur during lymphomagenesis.