A large number of tiny noncoding RNAs have been cloned and named microRNAs (miRs). Recently, we have reported that miR-15a and miR-16a, located at 13q14, are frequently deleted and͞or down-regulated in patients with B cell chronic lymphocytic leukemia, a disorder characterized by increased survival. To further investigate the possible involvement of miRs in human cancers on a genome-wide basis, we have mapped 186 miRs and compared their location to the location of previous reported nonrandom genetic alterations. Here, we show that miR genes are frequently located at fragile sites, as well as in minimal regions of loss of heterozygosity, minimal regions of amplification (minimal amplicons), or common breakpoint regions. Overall, 98 of 186 (52.5%) of miR genes are in cancer-associated genomic regions or in fragile sites. Moreover, by Northern blotting, we have shown that several miRs located in deleted regions have low levels of expression in cancer samples. These data provide a catalog of miR genes that may have roles in cancer and argue that the full complement of miRs in a genome may be extensively involved in cancers.

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...

Human adenocarcinomas commonly harbor mutations in the KRAS and MYC proto-oncogenes and the TP53 tumor suppressor gene. All three genetic lesions are potentially pro-angiogenic, as they sustain production of vascular endothelial growth factor (VEGF). Yet Kras-transformed mouse colonocytes lacking p53 formed indolent, poorly vascularized tumors, whereas additional transduction with a Myc-encoding retrovirus promoted vigorous vascularization and growth. In addition, VEGF levels were unaffected by Myc, but enhanced neovascularization correlated with downregulation of anti-angiogenic thrombospondin-1 (Tsp1) and related proteins, such as connective tissue growth factor (CTGF). Both Tsp1 and CTGF are predicted targets for repression by the miR-17-92 microRNA cluster, which was upregulated in colonocytes coexpressing K-Ras and c-Myc. Indeed, miR-17-92 knockdown with antisense 2'-O-methyl oligoribonucleotides partly restored Tsp1 and CTGF expression; in addition, transduction of Ras-only cells with a miR-17-92-encoding retrovirus reduced Tsp1 and CTGF levels. Notably, miR-17-92-transduced cells formed larger, better-perfused tumors. These findings establish a role for microRNAs in non-cell-autonomous Myc-induced tumor phenotypes.

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.

MicroRNAs (miRNAs) are a class of small noncoding RNA genes recently found to be abnormally expressed in several types of cancer. Here, we describe a recently developed methodology for miRNA gene expression profiling based on the development of a microchip containing oligonucleotides corresponding to 245 miRNAs from human and mouse genomes. We used these microarrays to obtain highly reproducible results that revealed tissuespecific miRNA expression signatures, data that were confirmed by assessment of expression by Northern blots, real-time RT-PCR, and literature search. The microchip oligolibrary can be expanded to include an increasing number of miRNAs discovered in various species and is useful for the analysis of normal and disease states.A recently discovered class of small noncoding RNAs, named microRNAs (miRNAs), has been identified in plants and animals (1, 2). The 19-to 22-nt active product is processed from a 60-to 110-nt pre-miRNA hairpin transcript thought to derive from a longer pre-miRNA product (3). It is believed that miRNAs act to regulate gene expression during development and differentiation, at the transcriptional and͞or translational level, although targets are still elusive (1, 4). We have previously shown frequent deletions and down-regulation of miR-15a and miR-16-1 miRNAs genes at 13q14 in B-cell chronic lymphocytic leukemia, the most common adult leukemia in the Western world (5). We also reported that human miRNA genes are frequently located at fragile sites and genomic regions involved in cancers (6), suggesting that the role of miRNA in human cancer may involve more than a few genes. In fact, two recent papers reported reduced accumulation of miR-145 and miR-143 in colorectal neoplasia (7) and high expression of precursor miRNA-155͞BIC RNA in children with Burkitt's lymphoma (8). Assessing cancer-specific expression levels for hundreds of miRNA genes is time consuming, and requires a large amount of total RNA (at least 20 g for each Northern blot) and autoradiographic techniques that require radioactive isotopes. To overcome these limitations and investigate alterations in expression of all known miRNAs in human cancer, we developed a miRNA microarray and established detection methodology for miRNA expression that overcomes the size limitation imposed by their length (18-22 nt). Methods miRNA Oligo Probe Design.A total of 281 miRNA precursor sequences (190 Homo sapiens, 88 Mus musculus, and 3 Arabidopsis thaliana) with annotated active sites were selected for oligonucleotide design. These correspond to human and mouse miRNAs found in the miRNA Registry (www.sanger.ac.uk͞Software͞Rfam͞ mirna; accessed June 2003) or collected from published papers (9-11). All of the sequences were confirmed by BLAST alignment with the corresponding genome at www.ncbi.nlm.nih.gov and the hairpin structures were analyzed at www.bioinfo.rpi.edu͞ applications͞mfold͞old͞rna. When two precursors with different length or slightly different base composition for the same miRNAs were found, both sequences were i...

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The basis of eukaryotic complexity is an intricate genetic architecture where parallel systems are involved in tuning gene expression, via RNA-DNA, RNA-RNA, RNA-protein, and DNA-protein interactions. In higher organisms, about 97% of the transcriptional output is represented by noncoding RNA (ncRNA) encompassing not only rRNA, tRNA, introns, 5¢ and 3¢ untranslated regions, transposable elements, and intergenic regions, but also a large, rapidly emerging family named microRNAs. MicroRNAs are short 20-22-nucleotide RNA molecules that have been shown to regulate the expression of other genes in a variety of eukaryotic systems. MicroRNAs are formed from larger transcripts that fold to produce hairpin structures and serve as substrates for the cytoplasmic Dicer, a member of the RNase III enzyme family. A recent analysis of the genomic location of human microRNA genes suggested that 50% of microRNA genes are located in cancer-associated genomic regions or in fragile sites. This review focuses on the possible implications of microRNAs in post-transcriptional gene regulation in mammalian diseases, with particular focus on cancer. We argue that developing mouse models for deleted and/or overexpressed microRNAs will be of invaluable interest to decipher the regulatory networks where microRNAs are involved.