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

MicroRNAs (miRNAs) represent a newly discovered class of posttranscriptional regulatory noncoding small RNAs that bind to targeted mRNAs and either block their translation or initiate their degradation. miRNA profiling of hematopoietic lineages in humans and mice showed that some miRNAs are differentially expressed during hematopoietic development, suggesting a role in hematopoietic cell differentiation. In addition, recent studies suggest the involvement of miRNAs in the initiation and progression of cancer. miR155 and BIC, its host gene, have been reported to accumulate in human B cell lymphomas, especially in diffuse large B cell lymphomas, Hodgkin lymphomas, and certain types of Burkitt lymphomas. Here, we show that E-mmu-miR155 transgenic mice exhibit initially a preleukemic pre-B cell proliferation evident in spleen and bone marrow, followed by frank B cell malignancy. These findings indicate that the role of miR155 is to induce polyclonal expansion, favoring the capture of secondary genetic changes for full transformation. Since their discovery in 1993 in Caenorhabditis elegans (1), there have been numerous reports that implicated these tiny molecules in the posttranscriptional regulation of a large array of proteins with very diverse roles, ranging from cell proliferation and differentiation to lipid metabolism (2-6).miRNA profiling of hematopoietic lineages in humans and mice showed that miRNAs are differentially expressed in the course of hematopoietic development, suggesting a potential role in hematopoietic differentiation (7-9). We have shown that miR-15a and miR-16-1 are deleted or down-regulated in Ϸ68% of cases of chronic lymphocytic leukemia (CLL) (10, 11), and that miRNAs genes are frequently located at fragile sites and other genomic regions involved in cancers (12). Transcripts of miR155 and BIC (its host gene) transcripts have been shown to accumulate in human B cell lymphomas, especially diffuse large B cell lymphomas (13), Hodgkin lymphomas (14), and subsets of Burkitt lymphomas (latency type III Epstein-Barr virus-positive Burkitt lymphoma; ref. 15). These reports provide indirect evidence that miR155 may play a role in B cell development and lymphomagenesis. We have also reported that miR155 is overexpressed in the aggressive form of CLL (11).Here, we show that the transgenic mice carrying a miR155 transgene whose expression is targeted to B cells (E-mmumiR155) exhibit initially a preleukemic pre-B cell proliferation, evident in spleen and bone marrow, and later develop a frank B cell malignancy. Results and DiscussionProduction and Characterization of E-mmu-miR155. We generated transgenic mice in which the expression of mmu-miR155 (mouse miR155) is under the control of a V H promoter-Ig heavy chain E enhancer, which becomes active at the late pro-B cell stage of the B cell development. Fifteen transgenic founders were identified by Southern blot hybridization, seven on C57BL͞B6 and eight on FVB͞N backgrounds. These were bred to wild-type mice of the same strain to produce 15 independent...

Progress in understanding the biology of multiple myeloma (MM), a plasma cell malignancy, has been slow. The discovery of microRNAs (miRNAs), a class of small noncoding RNAs targeting multiple mRNAs, has revealed a new level of gene expression regulation. To determine whether miRNAs play a role in the malignant transformation of plasma cells (PCs), we have used both miRNA microarrays and quantitative real time PCR to profile miRNA expression in MM-derived cell lines (n ‫؍‬ 49) and CD138؉ bone marrow PCs from subjects with MM (n ‫؍‬ 16), monoclonal gammopathy of undetermined significance (MGUS) (n ‫؍‬ 6), and normal donors (n ‫؍‬ 6). We identified overexpression of miR-21, miR-106bϳ25 cluster, miR181a and b in MM and MGUS samples with respect to healthy PCs. Selective up-regulation of miR-32 and miR-17ϳ92 cluster was identified in MM subjects and cell lines but not in MGUS subjects or healthy PCs. Furthermore, two miRNAs, miR-19a and 19b, that are part of the miR-17ϳ92 cluster, were shown to down regulate expression of SOCS-1, a gene frequently silenced in MM that plays a critical role as inhibitor of IL-6 growth signaling. We also identified p300-CBP-associated factor , a gene involved in p53 regulation, as a bona fide target of the miR106bϳ25 cluster, miR-181a and b, and miR-32. Xenograft studies using human MM cell lines treated with miR-19a and b, and miR-181a and b antagonists resulted in significant suppression of tumor growth in nude mice. In summary, we have described a MM miRNA signature, which includes miRNAs that modulate the expression of proteins critical to myeloma pathogenesis.PCAF ͉ SOCS-1 ͉ tumor suppressor gene ͉ MGUS ͉ plasma cells

MicroRNAs (miRNAs) are short noncoding RNAs regulating gene expression that play roles in human diseases, including cancer. Each miRNA is predicted to regulate hundreds of transcripts, but only few have experimental validation. In chronic lymphocytic leukemia (CLL), the most common adult human leukemia, miR-15a and miR-16-1 are lost or down-regulated in the majority of cases. After our previous work indicating a tumor suppressor function of miR-15a/16-1 by targeting the BCL2 oncogene, here, we produced a high-throughput profiling of genes modulated by miR-15a/16-1 in a leukemic cell line model (MEG-01) and in primary CLL samples. By combining experimental and bioinformatics data, we identified a miR-15a/16-1-gene signature in leukemic cells. Among the components of the miR-15a/ 16-1 signature, we observed a statistically significant enrichment in AU-rich elements (AREs). By examining the Gene Ontology (GO) database, a significant enrichment in cancer genes (such as MCL1, BCL2, ETS1, or JUN) that directly or indirectly affect apoptosis and cell cycle was found. (5), PicTar (6), and Diana microT (7) have been developed to identify miRNA targets, but only few of these predictions have been experimentally validated, supporting the rationale for a combination of bioinformatics and biological strategies to this aim. Two independent studies predicted that 20-30% of human genes could be controlled by miRNAs (8, 9). Deviations from normal miRNA expression patterns play roles in human diseases, including cancer (for reviews see refs. 10-15).The miR-15a/16-1 cluster resides at chromosome 13q14.3, a genomic region frequently deleted in B cell chronic lymphocytic leukemias (CLLs), and the two members of the cluster are cotranscribed and down-regulated in the majority of CLL patients (16). CLL is a disease with a frequent association in families (10-20% of patients have at least one first-degree relative with CLL) (17). Previously, we identified germ-line or somatic mutations in several miRNAs (including miR-16-1) in Ϸ15% of CLL patients, with the majority of the patients having a known personal or family history of CLL or other hematopoietic and solid tumors (18). These findings, together with the identification of an abnormal miR-15a/ 16-1 locus in the NZB strain of mice that naturally develop CLL (19), suggest that this cluster might play also a role in familial CLL.Among the targets of miR-15a and miR-16, we identified the antiapoptotic protein Bcl2, which is overexpressed in the malignant, mostly nondividing B cells of CLL (20), and in many solid and hematologic malignancies (21). Restoration of miR-15-a/16-1 induces apoptosis in MEG-01, a cell line derived from acute megakaryocytic leukemia (22). These data support a role for miR-15a and miR-16-1 as tumor-suppressor genes (TSGs) in CLLs and perhaps in other malignancies in which these genes are lost or down-regulated.Here, to investigate the mechanism of action of miR-15a and miR-16-1 as tumor suppressors in leukemias, we analyzed the effects of miR-15a and miR-16-1 on...