Micro-RNAs (miR genes) are a large family of highly conserved noncoding genes thought to be involved in temporal and tissuespecific gene regulation. MiRs are transcribed as short hairpin precursors (Ϸ70 nt) and are processed into active 21-to 22-nt RNAs by Dicer, a ribonuclease that recognizes target mRNAs via basepairing interactions. Here we show that miR15 and miR16 are located at chromosome 13q14, a region deleted in more than half of B cell chronic lymphocytic leukemias (B-CLL). Detailed deletion and expression analysis shows that miR15 and miR16 are located within a 30-kb region of loss in CLL, and that both genes are deleted or down-regulated in the majority (Ϸ68%) of CLL cases.

Chronic lymphocytic leukemia (CLL) is the most common human leukemia and is characterized by predominantly nondividing malignant B cells overexpressing the antiapoptotic B cell lymphoma 2 (Bcl2) protein. miR-15a and miR-16-1 are deleted or down-regulated in the majority of CLLs. Here, we demonstrate that miR-15a and miR-16-1 expression is inversely correlated to Bcl2 expression in CLL and that both microRNAs negatively regulate Bcl2 at a posttranscriptional level. BCL2 repression by these microRNAs induces apoptopsis in a leukemic cell line model. Therefore, miR-15 and miR-16 are natural antisense Bcl2 interactors that could be used for therapy of Bcl2-overexpressing tumors.

A unique microRNA signature is associated with prognostic factors and disease progression in CLL. Mutations in microRNA transcripts are common and may have functional importance.

M icroRNAs (miRNAs) represent a class of small, functional, noncoding RNAs of 19-23 nt cleaved from Ϸ60-to 110-nt hairpin precursors (1, 2). Hundreds of miRNAs have been identified in plants and animals. The miRNAs are involved in various biological processes, including cell proliferation and cell death during development, stress resistance, and fat metabolism, through the regulation of gene expression (3). Some miRNAs, such as miR-15a or miR-16-1 (4, 5), are widely expressed, whereas others, such as miR-1 in mammalian heart (6, 7) or miR-223 in granulocytes and macrophages (5), are expressed in a tissue-specific manner. Little else is known about miRNA expression patterns or function in normal or neoplastic cells.Understanding of the molecular pathogenesis of B cell chronic lymphocytic leukemia (CLL), the most common adult leukemia in the Western world, is incomplete. We have shown previously that miR-15a and miR-16-1 are located at chromosome 13q14.3 within a 30-kb region of loss in CLL cells and that both genes are deleted and͞or down-regulated in the majority of the analyzed CLL cell samples (4). These results provided the indication that deletion of miRNAs might be associated with a human malignancy. We also reported that 98 of the identified 186 miRNAs are located at fragile sites, minimal loss of heterozygosity regions, minimal regions of amplification, or common breakpoint regions in human cancers (8), suggesting that miRNAs might play a large and unanticipated role in the pathogenesis of human cancer. MethodsTissue Samples and CLL Samples. Forty-seven samples were used for this study, including 41 samples from 38 patients with CLL and 6 normal samples, including one lymph node, tonsillar CD5ϩ B cells from two normal donors, and blood mononuclear cells (MNC) from three normal donors. For three cases, two independent samples were collected and processed. CLL samples were obtained after informed consent from patients diagnosed with CLL at the CLL Research Consortium institutions. Briefly, blood was obtained from CLL patients, and MNC were isolated through Ficoll͞Hypaque gradient centrifugation (Amersham Pharmacia Biotech) and processed for RNA extraction according to described protocols (9). For the majority of samples, clinical and biological information, such as age at diagnosis, sex, Rai stage, presence͞absence of treatment, ZAP-70 expression, and IgV H gene mutation status were available (see Table 4, which is published as supporting information on the PNAS web site).Cell Preparation. MNC from peripheral blood of normal donors were separated by Ficoll-Hypaque density gradients. T cells were purified from these MNC by rosetting with neuraminidasetreated sheep erythrocyte and depletion of contaminant monocytes (Cd11bϩ); natural killer cells (CD16ϩ) and B lymphocytes (CD19ϩ) were purified by using magnetic beads (Dynabeads, Unipath, Milan) and specific mAbs (Becton Dickinson). Total B cells and CD5ϩ B cells were prepared from tonsillar lymphocytes as described (10). Briefly, tonsils were obtained from pat...

The most common human leukemia is B cell chronic lymphocytic leukemia (CLL), a malignancy of mature B cells with a characteristic clinical presentation but a variable clinical course. The rearranged immunoglobulin (Ig) genes of CLL cells may be either germ-line in sequence or somatically mutated. Lack of Ig mutations defined a distinctly worse prognostic group of CLL patients raising the possibility that CLL comprises two distinct diseases. Using genomic-scale gene expression profiling, we show that CLL is characterized by a common gene expression “signature,” irrespective of Ig mutational status, suggesting that CLL cases share a common mechanism of transformation and/or cell of origin. Nonetheless, the expression of hundreds of other genes correlated with the Ig mutational status, including many genes that are modulated in expression during mitogenic B cell receptor signaling. These genes were used to build a CLL subtype predictor that may help in the clinical classification of patients with this disease.

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Although the presence of an unmutated IgV(H) gene is strongly associated with the expression of ZAP-70, ZAP-70 is a stronger predictor of the need for treatment in B-cell CLL.

Noncoding RNA (ncRNA) transcripts are thought to be involved in human tumorigenesis. We report that a large fraction of genomic ultraconserved regions (UCRs) encode a particular set of ncRNAs whose expression is altered in human cancers. Genome-wide profiling revealed that UCRs have distinct signatures in human leukemias and carcinomas. UCRs are frequently located at fragile sites and genomic regions involved in cancers. We identified certain UCRs whose expression may be regulated by microRNAs abnormally expressed in human chronic lymphocytic leukemia, and we proved that the inhibition of an overexpressed UCR induces apoptosis in colon cancer cells. Our findings argue that ncRNAs and interaction between noncoding genes are involved in tumorigenesis to a greater extent than previously thought.