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.

MicroRNAs (miRNAs) are small noncoding RNAs, 19-24 nucleotides in length, that regulate gene expression and are expressed aberrantly in most types of cancer. MiRNAs also have been detected in the blood of cancer patients and can serve as circulating biomarkers. It has been shown that secreted miRNAs within exosomes can be transferred from cell to cell and can regulate gene expression in the receiving cells by canonical binding to their target messenger RNAs. Here we show that tumor-secreted miR-21 and miR-29a also can function by another mechanism, by binding as ligands to receptors of the Toll-like receptor (TLR) family, murine TLR7 and human TLR8, in immune cells, triggering a TLR-mediated prometastatic inflammatory response that ultimately may lead to tumor growth and metastasis. Thus, by acting as paracrine agonists of TLRs, secreted miRNAs are key regulators of the tumor microenvironment. This mechanism of action of miRNAs is implicated in tumor-immune system communication and is important in tumor growth and spread, thus representing a possible target for cancer treatment.icroRNAs (miRNAs) are small, noncoding RNAs, 19-24 nt in length, with gene-expression regulatory functions (1, 2) and are expressed aberrantly in most types of cancer (3, 4). MiRNAs also have been detected in the blood of cancer patients (5, 6) and can serve as circulating biomarkers (7). It has been shown that secreted miRNAs within exosomes can be transferred from cell to cell and can regulate gene expression in the receiving cells (8) by canonical binding to their target messenger RNAs (8, 9). More recently, it has been demonstrated that, in addition to their role as gene-expression regulators, miRNAs also directly interact with proteins (10).Members of the Toll-like receptor (TLR) family (namely, murine TLR7 and human TLR8) can recognize and bind viral single-stranded RNA (ssRNA) sequences on dendritic cells and B lymphocytes, leading to cell activation and cytokine production (11,12). TLRs are a family of receptors through which the mammalian innate immune system recognizes the presence of invading pathogens (13,14). Both murine TLR7 and human TLR8 bind to and are activated by 20-nt-long ssRNAs, which represent physiological ligands for these two receptors (12), located in intracellular endosomes. Circulating mature miRNAs are 19-24 nt in length and could represent tumor-released ligands of TLR7 and TLR8 involved in intercellular communication in the tumor microenvironment. Results and Discussion Identification of Specific miRNAs Released in Cancer Cell-DerivedExosomes. To identify which miRNAs are present in tumor-secreted exosomes, we isolated exosomes from the supernatant of A-549 and SK-MES lung cancer cell lines. First, we assessed the purified supernatant exosome fraction for enrichment in CD9 and CD63, two known exosome markers (SI Appendix, Fig. S1A) (8,15). By performing NanoString analysis, we observed that nine miRNAs (miR-16, -21, -27b, -29a, -133a, -193a-3p, -544, -563, and -1283) were present in exosomes derived from ...

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate expression of many genes. Recent studies suggest roles of miRNAs in carcinogenesis. We and others have shown that expression profiles of miRNAs are different in lung cancer vs. normal lung, although the significance of this aberrant expression is poorly understood. Among the reported down-regulated miRNAs in lung cancer, the miRNA (miR)-29 family (29a, 29b, and 29c) has intriguing complementarities to the 3-UTRs of DNA methyltransferase (DNMT)3A and -3B (de novo methyltransferases), two key enzymes involved in DNA methylation, that are frequently up-regulated in lung cancer and associated with poor prognosis. We investigated whether miR-29s could target DNMT3A and -B and whether restoration of miR-29s could normalize aberrant patterns of methylation in non-small-cell lung cancer. Here we show that expression of miR-29s is inversely correlated to DNMT3A and -3B in lung cancer tissues, and that miR-29s directly target both DNMT3A and -3B. The enforced expression of miR-29s in lung cancer cell lines restores normal patterns of DNA methylation, induces reexpression of methylation-silenced tumor suppressor genes, such as FHIT and WWOX, and inhibits tumorigenicity in vitro and in vivo. These findings support a role of miR-29s in epigenetic normalization of NSCLC, providing a rationale for the development of miRNA-based strategies for the treatment of lung cancer. epigenetics ͉ tumor-suppressor genes

Epithelial ovarian cancer (EOC) is the sixth most common cancer in women worldwide and, despite advances in detection and therapies, it still represents the most lethal gynecologic malignancy in the industrialized countries. Unfortunately, still relatively little is known about the molecular events that lead to the development of this highly aggressive disease. The relatively recent discovery of microRNAs (miRNA), a class of small noncoding RNAs targeting multiple mRNAs and triggering translation repression and/or RNA degradation, has revealed the existence of a new level of gene expression regulation. Multiple studies involving various types of human cancers proved that miRNAs have a causal role in tumorigenesis. Here we show that, in comparison to normal ovary, miRNAs are aberrantly expressed in human ovarian cancer. The overall miRNA expression could clearly separate normal versus cancer tissues. The most significantly overexpressed miRNAs were miR-200a, miR-141, miR-200c, and miR200b, whereas miR-199a, miR-140, miR-145, and miR-125b1 were among the most down-modulated miRNAs. We could also identify miRNAs whose expression was correlated with specific ovarian cancer biopathologic features, such as histotype, lymphovascular and organ invasion, and involvement of ovarian surface. Moreover, the levels of miR-21, miR-203, and miR-205, up-modulated in ovarian carcinomas compared with normal tissues, were significantly increased after 5-aza-2 ¶-deoxycytidine demethylating treatment of OVCAR3 cells, suggesting that the DNA hypomethylation could be the mechanism responsible for their overexpression. Our results indicate that miRNAs might play a role in the pathogenesis of human EOC and identify altered miRNA gene methylation as a possible epigenetic mechanism involved in their aberrant expression. [Cancer Res 2007;67(18):8699-707]

Pancreatic cancer may have a distinct miRNA expression pattern that may differentiate it from normal pancreas and chronic pancreatitis. miRNA expression patterns may be able to distinguish between long- and short-term survivors, but these findings need to be validated in other study populations.

Recent research has identified critical roles for microRNAs in a large number of cellular processes, including tumorigenic transformation. While significant progress has been made towards understanding the mechanisms of gene regulation by microRNAs, much less is known about factors affecting the expression of these noncoding transcripts. Here, we demonstrate for the first time a functional link between hypoxia, a welldocumented tumor microenvironment factor, and microRNA expression. Microarray-based expression profiles revealed that a specific spectrum of microRNAs (including miR-23, -24, -26, -27, -103, -107, -181, -210, and -213) is induced in response to low oxygen, at least some via a hypoxia-inducible-factor-dependent mechanism. Select members of this group (miR-26, -107, and -210) decrease proapoptotic signaling in a hypoxic environment, suggesting an impact of these transcripts on tumor formation. Interestingly, the vast majority of hypoxiainduced microRNAs are also overexpressed in a variety of human tumors.

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...