We report here that miR-155 and miR-125b play a role in innate immune response. LPS stimulation of mouse Raw 264.7 macrophages resulted in the up-regulation of miR-155 and down-regulation of miR-125b levels. The same changes also occurred when C57BL/6 mice were i.p. injected with LPS. Furthermore, the levels of miR-155 and miR-125b in Raw 264.7 cells displayed oscillatory changes in response to TNF-α. These changes were impaired by pretreating the cells with the proteasome inhibitor MG-132, suggesting that these two microRNAs (miRNAs) may be at least transiently under the direct control of NF-κB transcriptional activity. We show that miR-155 most probably directly targets transcript coding for several proteins involved in LPS signaling such as the Fas-associated death domain protein (FADD), IκB kinase ε (IKKε), and the receptor (TNFR superfamily)-interacting serine-threonine kinase 1 (Ripk1) while enhancing TNF-α translation. In contrast, miR-125b targets the 3′-untranslated region of TNF-α transcripts; therefore, its down-regulation in response to LPS may be required for proper TNF-α production. Finally, Eμ-miR-155 transgenic mice produced higher levels of TNF-α when exposed to LPS and were hypersensitive to LPS/d-galactosamine-induced septic shock. Altogether, our data suggest that the LPS/TNF-α-dependent regulation of miR-155 and miR-125b may be implicated in the response to endotoxin shock, thus offering new targets for drug design.

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...

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Summary Lung and liver cancers are among the most deadly types of cancer. Despite improvements in treatment over the past few decades, patient survival remains poor, underlining the need for development of targeted therapies. MicroRNAs represent a class of small RNAs, frequently deregulated in human malignancies. We now report that miR221&222 are over-expressed in aggressive non small cell lung cancer and hepatocarcinoma cells, as compared with less invasive and/or normal lung and liver cells. We show that miR-221&222, by targeting PTEN and TIMP3 tumor suppressors, induce TRAIL resistance and enhance cellular migration through the activation of the AKT pathway and metallopeptidases. Finally, we demonstrate that the MET oncogene is involved in miR-221&222 activation, through the c-Jun transcription factor.

The human genome is replete with long non-coding RNAs (lncRNA), many of which are transcribed and likely to have a functional role. Microarray analysis of > 23 000 lncRNAs revealed downregulation of 712 (~3%) lncRNA in malignant hepatocytes, among which maternally expressed gene 3 (MEG3) was downregulated by 210-fold relative to expression in non-malignant hepatocytes. MEG3 expression was markedly reduced in four human hepatocellular cancer (HCC) cell lines compared with normal hepatocytes by real-time PCR. RNA in situ hybridization showed intense cytoplasmic expression of MEG3 in non-neoplastic liver with absent or very weak expression in HCC tissues. Enforced expression of MEG3 in HCC cells significantly decreased both anchorage-dependent and -independent cell growth, and induced apoptosis. MEG3 promoter hypermethylation was identified by methylation-specific PCR and MEG3 expression was increased with inhibition of methylation with either 5-Aza-2-Deoxycytidine, or siRNA to DNA Methyltransferase (DNMT) 1 and 3b in HCC cells. MiRNA-dependent regulation of MEG3 expression was studied by evaluating the involvement of miR-29, which can modulate DNMT 1 and 3. Overexpression of mir-29a increased expression of MEG3. GTL2, the murine homolog of MEG3, was reduced in liver tissues from hepatocyte-specific miR-29a/b1 knock-out mice compared with wild-type controls. These data show that methylation-dependent tissue-specific regulation of the lncRNA MEG3 by miR-29a may contribute to HCC growth and highlight the inter-relationship between two classes of non-coding RNA, miRNAs and lncRNAs, and epigenetic regulation of gene expression.

The WW domain-containing oxidoreductase (WWOX) spans the second most common fragile site of the human genome, FRA16D, located at 16q23, and its expression is altered in several types of human cancer. We have previously shown that restoration of WWOX expression in cancer cells suppresses tumorigenicity. common fragile site ͉ FHIT ͉ knockout ͉ osteosarcoma ͉ lung cancer T he WW domain-containing oxidoreductase (WWOX) encodes a 46-kDa protein that contains two WW domains and a short-chain dehydrogenase/reductase domain (1-3). The WWOX gene is altered by deletions or translocations in a large fraction of many cancer types including breast, prostate, esophageal, lung, stomach, and pancreatic carcinomas (2, 4-9). WWOX protein is lost or reduced in the majority of these cancers and in a large fraction of other cancer types (10, 11). WWOX spans the second most active common fragile sites, FRA16D (reviewed in ref. 12). Common fragile sites are large regions of profound genomic instability found in all individuals. Following partial inhibition of DNA synthesis, those regions show site-specific gaps or breaks on metaphase chromosomes. In addition, common fragile sites exhibit induction of sister chromatid exchange and show a high rate of translocations and deletions in somatic cell hybrids (13). Because FRA16D maps within regions of frequent loss of heterozygosity, and is associated with homozygous deletions in various adenocarcinomas and with chromosomal translocations in multiple myeloma (2), these rearrangements have been suggested to inactivate the WWOX gene. On the other hand, ectopic overexpression of WWOX in cancer cells lacking expression of endogenous WWOX results in significant growth inhibition and prevents the development of tumors in athymic nude mice (14,15). In addition, we reported that restoration of WWOX expression in cancer cells results in caspase-mediated apoptosis (15). Thus, these data suggest that WWOX may act as a tumor suppressor Biochemical and functional characterization of WWOX has shown that it interacts with proline-tyrosine rich motifcontaining proteins. WWOX associates via its first WW domain with p73 and enhances p73-mediated apoptosis (16). WWOX also binds to the PPPY motif of AP2␥ and ErbB4, established oncogenes in breast cancer (17,18). Interestingly, WWOX suppresses the transcriptional ability of these target proteins by sequestering them in the cytoplasm (16)(17)(18). Taken together, accumulating evidence, both in cell culture and in nude mice, suggests that WWOX functions as a tumor suppressor, although no direct in vivo proof has yet been reported to verify WWOX as a bona fide tumor suppressor. To define the role of WWOX protein in cancer development, we generated mice carrying a targeted deletion of the Wwox gene. The murine Wwox locus is similar to its human homolog (19), spans the Fra8E1 common fragile site, and is highly conserved. Here, we demonstrate that the loss of both alleles of Wwox results in osteosarcomas in some early postnatal mice, whereas loss of one allele signifi...

We studied miRNA profiles in 4419 human samples (3312 neoplastic, 1107 nonmalignant), corresponding to 50 normal tissues and 51 cancer types. The complexity of our database enabled us to perform a detailed analysis of microRNA (miRNA) activities. We inferred genetic networks from miRNA expression in normal tissues and cancer. We also built, for the first time, specialized miRNA networks for solid tumors and leukemias. Nonmalignant tissues and cancer networks displayed a change in hubs, the most connected miRNAs. hsa-miR-103/106 were downgraded in cancer, whereas hsa-miR-30 became most prominent. Cancer networks appeared as built from disjointed subnetworks, as opposed to normal tissues. A comparison of these nets allowed us to identify key miRNA cliques in cancer. We also investigated miRNA copy number alterations in 744 cancer samples, at a resolution of 150 kb. Members of miRNA families should be similarly deleted or amplified, since they repress the same cellular targets and are thus expected to have similar impacts on oncogenesis. We correctly identified hsa-miR-17/92 family as amplified and the hsa-miR-143/145 cluster as deleted. Other miRNAs, such as hsa-miR-30 and hsa-miR-204, were found to be physically altered at the DNA copy number level as well. By combining differential expression, genetic networks, and DNA copy number alterations, we confirmed, or discovered, miRNAs with comprehensive roles in cancer. Finally, we experimentally validated the miRNA network with acute lymphocytic leukemia originated in Mir155 transgenic mice. Most of miRNAs deregulated in these transgenic mice were located close to hsa-miR-155 in the cancer network