An Atlas of the MicroRNA Genome in Human DLBCL.

Abstract

MicroRNAs (miRNA) preferentially map to unstable chromosomal regions suggesting that structural defects may contribute to their role in cancer. Diffuse large B-cell lymphoma (DLBCL) is characterized by recurring chromosomal translocations and DNA imbalances, but the integrity of the miRNA genome in this neoplasm is unknown. To address this issue at a genome-wide level we designed a novel oligonucleotide-based array-CGH platform (Agilent) that covers at high density (∼500bp interval) the loci for 471 human miRNAs and 17 genes involved in miRNA biogenesis. We used this platform to analyze the miRNA genes of a collection of 87 well-characterized DLBCLs, including 60 primary tumors and 27 DLBCL cell lines. We found widespread disruption of miRNA genes in these lymphomas with 57 of the 60 primary tumors (95%) and 100% of the cell lines exhibiting alteration of at least one miRNA locus. Only 8 miRNA loci were intact in all samples. Most of the tumors had multiple miRNA loci disrupted, a phenomenon reminiscent of the multiple and cooperative aberrations of classical mRNA genes in cancer. The majority of the frequently disrupted miRNAs exhibited gains rather than loss of genetic material. A total of 48 miRNAs (representing 21 independent miRNA clusters) showed gains of genetic material in more than 20% of the cases, whereas only seven miRNA genes had loss of material in a significant percentage of the tumors. The five miRNA loci that most frequently exhibited DNA gain included miR-511-1, miR-339, miR-1-1, miR-133a-2 and miR-589, and the miRNA genes that most commonly exhibited loss of material were miR-31, miR-548a-2, miR-491, and the miR-15a/miR-16 cluster. In contrast, the genes involved in miRNA biogenesis were highly stable and only AGO2, DGCR8-Pasha and FMR1 were disrupted in more 10% of the cases. There was significant overlap between the aberrations found in primary tumors and DLBCL cell lines, especially concerning the miRNAs targeted for deletion. An examination of eight miRNAs previously found to be expressed and/or play a role in B-cell biology (miRs 142, 144, 150, 155, 223, 26a, 191 and 16) revealed that most of these genes exhibited frequent gains of genetic material, with the exception of a highly stable miR-155 locus and the miR-16 locus on chromosome 13q, which showed loss of genetic material in ∼30% of the cases. These data indicated that the well known over-expression of miR-155 in DLBCL is not linked to copy number changes and suggested that genomic instability may lead to abnormal expression of the other B-cell relevant miRNAs. To test this hypothesis we determined the expression of miR-26a and miR-16, the two B-cell relevant miRNAs with the highest frequency of genetic alterations, using a real-time quantitative RT-PCR assay that specifically amplifies the mature miRNA. Surprisingly, there was no significant difference in the expression of miR-16 in tumors or cell lines with or without deletion of genetic material at this locus, suggesting that this miRNA may not play a role in DLBCL. In contrast, miR-26a expression correlated well with its copy number and was ∼3.5 higher in tumors (p< 0.01) and cell lines (p=0.02) with DNA gain than in those with an intact genomic locus. These data suggest that mirR-26a, which is predicted to modulate PTEN expression, may act as an oncogene in DLBCL. In summary, our high resolution analysis creates a map of miRNA integrity in DLBCL, a critical tool to start to delineate their role in this tumor.