Showing papers by "Carlo M. Croce published in 2008"

MicroRNA expression profiles associated with prognosis and therapeutic outcome in colon adenocarcinoma

Abstract: COLON ADENOCARCINOMA IS A major cause of cancer mortality worldwide1 Colorectal cancer is the third most common and second leading cause of cancer death in the United States2 Even though 5-year mortality rates have modestly declined over the last 3 decades,3 there is still a need to identify new prognostic biomarkers and therapeutic targets for this disease Currently, chemotherapy has significant therapeutic value, but surgery is the only curative form of treatment4 Ideal therapeutic targets should be causally associated with disease and amenable to designing therapeutic interventions, whereas ideal biomarkers should be easy to measure and have strong associations with clinical outcomes MicroRNAs could match both criteria5-8 MicroRNAs are 18- to 25-nucleotide, noncoding RNA molecules that regulate the translation of many genes9 Since their discovery,10,11 microRNAs have been found to regulate a variety of cellular processes including apoptosis,12-14 differentiation,10,11,15 and cell proliferation16 MicroRNAs may also have a causal role in carcinogenesis5,17,18 MicroRNA expression levels are altered in most tumor types,19,20 including colon tumors20-23 Experimental manipulation of specific microRNAs modulates tumor development in mouse-model systems16,24-26 The prognostic potential of microRNAs has also been demonstrated for chronic lymphocytic leukemia,6 lung cancer,7 pancreatic cancer,27 and neuroblastomas28 If aberrant microRNA expression is causal to carcinogenesis, inhibiting specific microRNAs may have therapeutic implications Modified antisense oligonucleotides can easily be designed to specifically inhibit microRNA function29 Antagomirs are one type of antisense oligonucleotide that has proven effective at inhibiting microRNA function in vivo in mice30 The ease of designing specific inhibitors of microRNA function makes them candidates for therapeutic targets Given the therapeutic and prognostic potential for microRNAs in cancer, we evaluated microRNA profiles of colon tumors and paired nontumorous tissue to study their potential role in tumor formation, diagnosis, and response to chemotherapy in colon carcinoma

MicroRNA Signatures in Human Ovarian Cancer

Abstract: The present invention provides novel methods for the diagnosis of ovarian cancer using at least one miR selected from miR-200b, miR-141, miR-199a, miR-140, miR-145 and miR-125b1miR-200c. The invention also provides methods of identifying anti-ovarian cancer agents and a kit for detecting ovarian cancer.

A microRNA DNA methylation signature for human cancer metastasis

Abstract: MicroRNAs (miRNAs) are small, noncoding RNAs that can contribute to cancer development and progression by acting as oncogenes or tumor suppressor genes. Recent studies have also linked different sets of miRNAs to metastasis through either the promotion or suppression of this malignant process. Interestingly, epigenetic silencing of miRNAs with tumor suppressor features by CpG island hypermethylation is also emerging as a common hallmark of human tumors. Thus, we wondered whether there was a miRNA hypermethylation profile characteristic of human metastasis. We used a pharmacological and genomic approach to reveal this aberrant epigenetic silencing program by treating lymph node metastatic cancer cells with a DNA demethylating agent followed by hybridization to an expression microarray. Among the miRNAs that were reactivated upon drug treatment, miR-148a, miR-34b/c, and miR-9 were found to undergo specific hypermethylation-associated silencing in cancer cells compared with normal tissues. The reintroduction of miR-148a and miR-34b/c in cancer cells with epigenetic inactivation inhibited their motility, reduced tumor growth, and inhibited metastasis formation in xenograft models, with an associated down-regulation of the miRNA oncogenic target genes, such as C-MYC, E2F3, CDK6, and TGIF2. Most important, the involvement of miR-148a, miR-34b/c, and miR-9 hypermethylation in metastasis formation was also suggested in human primary malignancies (n = 207) because it was significantly associated with the appearance of lymph node metastasis. Our findings indicate that DNA methylation-associated silencing of tumor suppressor miRNAs contributes to the development of human cancer metastasis.

Oncogenes and cancer.

Abstract: This review — the first in a series concerning the biology of cancer — is a comprehensive survey of oncogenes, tumor-suppressor genes, and microRNA genes in cancer cells. Work on the recently discovered microRNA genes in malignant cells has revealed new complexities in the regulation of oncogenes and tumor-suppressor genes and new opportunities for the treatment of cancer.

MiR-15a and miR-16-1 cluster functions in human leukemia.

Abstract: 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.

Genomic profiling of microRNA and messenger RNA reveals deregulated microRNA expression in prostate cancer.

Abstract: MicroRNAs are small non-coding RNAs that regulate the expression of protein-coding genes. To evaluate the involvement of microRNAs in prostate cancer, we determined genome-wide expression of microRNAs and mRNAs in 60 primary prostate tumors and 16 non-tumor prostate tissues. The mRNA analysis revealed that key components of microRNA processing and several microRNA host genes, e.g., MCM7 and C9orf5, were significantly up-regulated in prostate tumors. Consistent with these findings, tumors expressed the miR-106b-25 cluster, which maps to intron 13 of MCM7, and miR-32, which maps to intron 14 of C9orf5, at significantly higher levels than non-tumor prostate. The expression levels of other microRNAs, including a number of miR-106b-25 cluster homologues, were also altered in prostate tumors. Additional differences in microRNA abundance were found between organ-confined tumors and those with extraprostatic disease extension. Lastly, we found evidence that some microRNAs are androgen-regulated and that tumor microRNAs influence transcript abundance of protein-coding target genes in the cancerous prostate. In cell culture, E2F1 and p21/WAF1 were identified as targets of miR-106b, Bim of miR-32, and exportin-6 and protein tyrosine kinase 9 of miR-1. In summary, microRNA expression becomes altered with the development and progression of prostate cancer. Some of these microRNAs regulate the expression of cancer-related genes in prostate cancer cells.

MicroRNAs regulate critical genes associated with multiple myeloma pathogenesis

Abstract: 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-106b approximately 25 cluster, miR-181a and b in MM and MGUS samples with respect to healthy PCs. Selective up-regulation of miR-32 and miR-17 approximately 92 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-17 approximately 92 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 miR106b approximately 25 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.

Genomic and epigenetic alterations deregulate microRNA expression in human epithelial ovarian cancer

Abstract: MicroRNAs (miRNAs) are an abundant class of small noncoding RNAs that function as negative gene regulators. miRNA deregulation is involved in the initiation and progression of human cancer; however, the underlying mechanism and its contributions to genome-wide transcriptional changes in cancer are still largely unknown. We studied miRNA deregulation in human epithelial ovarian cancer by integrative genomic approach, including miRNA microarray (n = 106), array-based comparative genomic hybridization (n = 109), cDNA microarray (n = 76), and tissue array (n = 504). miRNA expression is markedly down-regulated in malignant transformation and tumor progression. Genomic copy number loss and epigenetic silencing, respectively, may account for the down-regulation of ≈15% and at least ≈36% of miRNAs in advanced ovarian tumors and miRNA down-regulation contributes to a genome-wide transcriptional deregulation. Last, eight miRNAs located in the chromosome 14 miRNA cluster (Dlk1-Gtl2 domain) were identified as potential tumor suppressor genes. Therefore, our results suggest that miRNAs may offer new biomarkers and therapeutic targets in epithelial ovarian cancer.

Distinctive microRNA signature of acute myeloid leukemia bearing cytoplasmic mutated nucleophosmin

Abstract: Acute myeloid leukemia (AML) carrying NPM1 mutations and cytoplasmic nucleophosmin (NPMc+ AML) accounts for about one-third of adult AML and shows distinct features, including a unique gene expression profile. MicroRNAs (miRNAs) are small noncoding RNAs of 19–25 nucleotides in length that have been linked to the development of cancer. Here, we investigated the role of miRNAs in the biology of NPMc+ AML. The miRNA expression was evaluated in 85 adult de novo AML patients characterized for subcellular localization/mutation status of NPM1 and FLT3 mutations using a custom microarray platform. Data were analyzed by using univariate t test within BRB tools. We identified a strong miRNA signature that distinguishes NPMc+ mutated (n = 55) from the cytoplasmic-negative (NPM1 unmutated) cases (n = 30) and includes the up-regulation of miR-10a, miR-10b, several let-7 and miR-29 family members. Many of the down-regulated miRNAs including miR-204 and miR-128a are predicted to target several HOX genes. Indeed, we confirmed that miR-204 targets HOXA10 and MEIS1, suggesting that the HOX up-regulation observed in NPMc+ AML may be due in part by loss of HOX regulators-miRNAs. FLT3-ITD+ samples were characterized by up-regulation of miR-155. Further experiments demonstrated that the up-regulation of miR-155 was independent from FLT3 signaling. Our results identify a unique miRNA signature associated with NPMc+ AML and provide evidence that support a role for miRNAs in the regulation of HOX genes in this leukemia subtype. Moreover, we found that miR-155 was strongly but independently associated with FLT3-ITD mutations.

A microRNA signature for a BMP2-induced osteoblast lineage commitment program.

Abstract: Bone morphogenetic proteins (BMPs) are potent morphogens that activate transcriptional programs for lineage determination. How BMP induction of a phenotype is coordinated with microRNAs (miRNAs) that inhibit biological pathways to control cell differentiation, remains unknown. Here, we show by profiling miRNAs during BMP2 induced osteogenesis of C2C12 mesenchymal cells, that 22 of 25 miRNAs which significantly changed in response to BMP2 are down-regulated. These miRNAs are each predicted to target components of multiple osteogenic pathways. We characterize two representative miRNAs and show that miR-133 directly targets Runx2, an early BMP response gene essential for bone formation, and miR-135 targets Smad5, a key transducer of the BMP2 osteogenic signal, controlled through their 3′UTR sequences. Both miRNAs functionally inhibit differentiation of osteoprogenitors by attenuating Runx2 and Smad5 pathways that synergistically contribute to bone formation. Although miR-133 is known to promote MEF-2-dependent myogenesis, we have identified a second complementary function to inhibit Runx2-mediated osteogenesis. Our key finding is that BMP2 controls bone cell determination by inducing miRNAs that target muscle genes but mainly by down-regulating multiple miRNAs that constitute an osteogenic program, thereby releasing from inhibition pathway components required for cell lineage commitment. Thus, our studies establish a mechanism for BMP morphogens to selectively induce a tissue-specific phenotype and suppress alternative lineages.

MicroRNA expression in cytogenetically normal acute myeloid leukemia

Abstract: Background A role of microRNAs in cancer has recently been recognized. However, little is known about the role of microRNAs in acute myeloid leukemia (AML). Methods Using microRNA expression profiling, we studied samples of leukemia cells from adults under the age of 60 years who had cytogenetically normal AML and high-risk molecular features — that is, an internal tandem duplication in the fms-related tyrosine kinase 3 gene (FLT3–ITD), a wild-type nucleophosmin (NPM1), or both. A microRNA signature that was associated with event-free survival was derived from a training group of 64 patients and tested in a validation group of 55 patients. For the latter, a microRNA compound covariate predictor (called a microRNA summary value) was computed on the basis of weighted levels of the microRNAs forming the outcome signature. Results Of 305 microRNA probes, 12 (including 5 representing microRNA-181 family members) were associated with event-free survival in the training group (P<0.005). In the validation group, ...

Methylation mediated silencing of MicroRNA-1 gene and its role in hepatocellular carcinogenesis.

Abstract: MicroRNAs (miR) are a class of small (∼21 nucleotide) noncoding RNAs that, in general, negatively regulate gene expression. Some miRs harboring CGIs undergo methylation-mediated silencing, a characteristic of many tumor suppressor genes. To identify such miRs in liver cancer, the miRNA expression profile was analyzed in hepatocellular carcinoma (HCC) cell lines treated with 5-azacytidine (DNA hypomethylating agent) and/or trichostatin A (histone deacetylase inhibitor). The results showed that these epigenetic drugs differentially regulate expression of a few miRs, particularly miR-1-1 , in HCC cells. The CGI spanning exon 1 and intron 1 of miR-1-1 was methylated in HCC cell lines and in primary human HCCs but not in matching liver tissues. The miR-1-1 gene was hypomethylated and activated in DNMT1 −/− HCT 116 cells but not in DNMT3B null cells, indicating a key role for DNMT1 in its methylation. miR-1 expression was also markedly reduced in primary human hepatocellular carcinomas compared with matching normal liver tissues. Ectopic expression of miR-1 in HCC cells inhibited cell growth and reduced replication potential and clonogenic survival. The expression of FoxP1 and MET harboring three and two miR-1 cognate sites, respectively, in their respective 3′-untranslated regions, was markedly reduced by ectopic miR-1. Up-regulation of several miR-1 targets including FoxP1, MET, and HDAC4 in primary human HCCs and down-regulation of their expression in 5-AzaC–treated HCC cells suggest their role in hepatocarcinogenesis. The inhibition of cell cycle progression and induction of apoptosis after re-expression of miR-1 are some of the mechanisms by which DNA hypomethylating agents suppress hepatocarcinoma cell growth. [Cancer Res 2008;68(13):5049–58]

Emerging Role of miR-106b-25/miR-17-92 Clusters in the Control of Transforming Growth Factor β Signaling

Abstract: Inactivation of the transforming growth factor β (TGFβ) tumor suppressor pathway is a main step in the development of a variety of human tumors. The miR-106b-25 and miR-17-92 clusters are emerging as key modulators of TGFβ signaling in gastrointestinal and other tumors, interfering with cell cycle arrest and apoptosis when overexpressed in cancer cells. Genetic ablation of these microRNAs (miRNAs) reveals their physiologic role in the control of liver and central nervous system apoptosis, supporting the notion that miRNA-based homeostatic mechanisms can be usurped by cancer cells to resist TGFβ tumor suppression. [Cancer Res 2008;68(20):8191–4]

MicroRNAs modulate the chemosensitivity of tumor cells

Abstract: MicroRNAs are strongly implicated in such processes as development, carcinogenesis, cell survival, and apoptosis. It is likely, therefore, that they can also modulate sensitivity and resistance to anticancer drugs in substantial ways. To test this hypothesis, we studied the pharmacologic roles of three microRNAs previously implicated in cancer biology (let-7i, mir-16, and mir-21) and also used in silico methods to test pharmacologic microRNA effects more broadly. In the experimental system, we increased the expression of individual microRNAs by transfecting their precursors (which are active) or suppressed the expression by transfection of antisense oligomers. In three NCI-60 human cancer cell lines, a panel of 60 lines used for anticancer drug discovery, we assessed the growth-inhibitory potencies of 14 structurally diverse compounds with known anticancer activities. Changing the cellular levels of let-7i, mir-16, and mir-21 affected the potencies of a number of the anticancer agents by up to 4-fold. The effect was most prominent with mir-21, with 10 of 28 cell-compound pairs showing significant shifts in growth-inhibitory activity. Varying mir-21 levels changed potencies in opposite directions depending on compound class; indicating that different mechanisms determine toxic and protective effects. In silico comparison of drug potencies with microRNA expression profiles across the entire NCI-60 panel revealed that approximately 30 microRNAs, including mir-21, show highly significant correlations with numerous anticancer agents. Ten of those microRNAs have already been implicated in cancer biology. Our results support a substantial role for microRNAs in anticancer drug response, suggesting novel potential approaches to the improvement of chemotherapy.

MicroRNA Microarray Identifies Let-7i as a Novel Biomarker and Therapeutic Target in Human Epithelial Ovarian Cancer

Abstract: MicroRNAs (miRNA) are approximately 22-nucleotide noncoding RNAs that negatively regulate protein-coding gene expression in a sequence-specific manner via translational inhibition or mRNA degradation. Our recent studies showed that miRNAs exhibit genomic alterations at a high frequency and their expression is remarkably deregulated in ovarian cancer, strongly suggesting that miRNAs are involved in the initiation and progression of this disease. In the present study, we performed miRNA microarray to identify the miRNAs associated with chemotherapy response in ovarian cancer and found that let-7i expression was significantly reduced in chemotherapy-resistant patients (n = 69, P = 0.003). This result was further validated by stem-loop real-time reverse transcription-PCR (n = 62, P = 0.015). Both loss-of-function (by synthetic let-7i inhibitor) and gain-of-function (by retroviral overexpression of let-7i) studies showed that reduced let-7i expression significantly increased the resistance of ovarian and breast cancer cells to the chemotherapy drug, cis-platinum. Finally, using miRNA microarray, we found that decreased let-7i expression was significantly associated with the shorter progression-free survival of patients with late-stage ovarian cancer (n = 72, P = 0.042). This finding was further validated in the same sample set by stem-loop real-time reverse transcription-PCR (n = 62, P = 0.001) and in an independent sample set by in situ hybridization (n = 53, P = 0.049). Taken together, our results strongly suggest that let-7i might be used as a therapeutic target to modulate platinum-based chemotherapy and as a biomarker to predict chemotherapy response and survival in patients with ovarian cancer.

MicroRNA signatures of TRAIL resistance in human non-small cell lung cancer.

Abstract: To define novel pathways that regulate susceptibility to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in non-small cell lung cancer (NSCLC), we have performed genome-wide expression profiling of microRNAs (miRs). We show that in TRAIL-resistant NSCLC cells, levels of different miRs are increased, and in particular, miR-221 and -222. We demonstrate that these miRs impair TRAIL-dependent apoptosis by inhibiting the expression of key functional proteins. Indeed, transfection with anti-miR-221 and -222 rendered CALU-1-resistant cells sensitive to TRAIL. Conversely, H460-sensitive cells treated with -221 and -222 pre-miRs become resistant to TRAIL. miR-221 and -222 target the 3'-UTR of Kit and p27(kip1) mRNAs, but interfere with TRAIL signaling mainly through p27(kip1). In conclusion, we show that high expression levels of miR-221 and -222 are needed to maintain the TRAIL-resistant phenotype, thus making these miRs as promising therapeutic targets or diagnostic tool for TRAIL resistance in NSCLC.

MicroRNA involvement in hepatocellular carcinoma

Abstract: Hepatocellular carcinoma (HCC) is the third cause of cancer-related death worldwide. Curative options for HCC are limited and exclusively available for patients carrying an early stage HCC. In advanced stages, traditional chemotherapy proved to be only marginally effective or even toxic. Thus, the identification of new treatment options is needed. New targets for non-conventional treatment will necessarily take advantage of progresses on the molecular pathogenesis of HCC. MicroRNAs (miRNAs) are a group of tiny RNAs with a fundamental role in the regulation of gene expression. Aberrant expression of several miRNAs was found to be involved in human hepatocarcinogenesis. miRNA expression signatures were correlated with bio-pathological and clinical features of HCC. In some cases, aberrantly expressed miRNAs could be linked to cancer-associated pathways, indicating a direct role in liver tumourigenesis. For example, up-regulation of mir-221 and mir-21 could promote cell cycle progression, reduce cell death and favour angiogenesis and invasion. These findings suggest that miRNAs could become novel molecular targets for HCC treatment. The demonstration of in vivo efficacy and safety of anti-miRNA compounds has opened the way to their use in clinical trials.

Mechanisms of microRNA deregulation in human cancer.

Abstract: microRNAs (miRNAs) are an abundant class of small non-coding RNAs that function as gene regulators. Although deregulation of miRNA expression is involved in the initiation and progression of tumorigenesis, the underlying mechanisms of miRNA deregulation in human cancer are still largely unknown. Increasing evidence indicates that transcriptional deregulations, epigenetic alterations, mutations, DNA copy number abnormalities and defects in the miRNA biogenesis machinery might contribute to miRNA deregulation in human cancer. A clearer understanding of the mechanisms involved in miRNA deregulation in human cancer will contribute greatly to the development of new miRNA-based strategies in cancer diagnosis and treatment.

MicroRNAs 221 and 222 Bypass Quiescence and Compromise Cell Survival

Abstract: MicroRNAs (miRNA) have tumor suppressive and oncogenic potential in human cancer, but whether and how miRNAs control cell cycle progression is not understood. To address this question, we carried out a comprehensive analysis of miRNA expression during serum stimulation of quiescent human cells. Time course analyses revealed that four miRNAs are up-regulated and >100 miRNAs are down-regulated, as cells progress beyond the G1-S phase transition. We analyzed the function of two up-regulated miRNAs (miR-221 and miR-222) that are both predicted to target the cell growth suppressive cyclin-dependent kinase inhibitors p27 and p57. Our results show that miR-221 and miR-222 both directly target the 3′ untranslated regions of p27 and p57 mRNAs to reduce reporter gene expression, as well as diminish p27 and p57 protein levels. Functional studies show that miR-221 and miR-222 prevent quiescence when elevated during growth factor deprivation and induce precocious S-phase entry, thereby triggering cell death. Thus, the physiologic up-regulation of miR-221 and miR-222 is tightly linked to a cell cycle checkpoint that ensures cell survival by coordinating competency for initiation of S phase with growth factor signaling pathways that stimulate cell proliferation. [Cancer Res 2008;68(8):2773–80]

MicroRNA expression profiling using microarrays

Abstract: Microarray technology is a powerful high-throughput tool capable of monitoring the expression of thousands of small noncoding RNAs at once within tens of samples processed in parallel in a single experiment. To conduct a genome-wide analysis of miRNA expression of normal and disease samples, such as cancer, and to distinguish expression signatures associated with diagnosis, prognosis and therapeutic interventions, we have developed a unique miRNA microarray assay on a CodeLink platform. The miRNA array consists of 4,104 probes printed in duplicate. This array can simultaneously profile more than 1,500 mature miRNAs and their corresponding precursors from 474 human and 373 mouse miRNA genes. The full protocol details of the miRNA microarray assay developed by our group are described here, including miRNA oligo probe design, array fabrication and miRNA target preparation (by reverse transcription of total RNA), target-probe hybridization on array, signal detection and data analysis. The assay is simple, can be easily standardized and allows the reproducible profiling of up to 24 total RNA samples within 24 h.

Aberrant chromatin at genes encoding stem cell regulators in human mixed-lineage leukemia

Abstract: Mixed-lineage leukemia (MLL) fusion proteins are potent inducers of leukemia, but how these proteins generate aberrant gene expression programs is poorly understood. Here we show that the MLL-AF4 fusion protein occupies developmental regulatory genes important for hematopoietic stem cell identity and self-renewal in human leukemia cells. These MLL-AF4-bound regions have grossly altered chromatin structure, with histone modifications catalyzed by trithorax group proteins and DOT1 extending across large domains. Our results define direct targets of the MLL fusion protein, reveal the global role of epigenetic misregulation in leukemia, and identify new targets for therapeutic intervention in cancer.

MicroRNAs in normal and malignant hematopoiesis.

Abstract: Purpose of reviewThe discovery of a novel class of gene regulators, named microRNAs, has changed the landscape of human genetics. In hematopoiesis, recent work has improved our understanding of the role of microRNAs in hematopoietic differentiation and leukemogenesis.Recent findingsUsing animal mode

miR-181b negatively regulates activation-induced cytidine deaminase in B cells

Abstract: Activated B cells reshape their primary antibody repertoire after antigen encounter by two molecular mechanisms: somatic hypermutation (SHM) and class switch recombination (CSR). SHM and CSR are initiated by activation-induced cytidine deaminase (AID) through the deamination of cytosine residues on the immunoglobulin loci, which leads to the generation of DNA mutations or double-strand break intermediates. As a bystander effect, endogenous AID levels can also promote the generation of chromosome translocations, suggesting that the fine tuning of AID expression may be critical to restrict B cell lymphomagenesis. To determine whether microRNAs (miRNAs) play a role in the regulation of AID expression, we performed a functional screening of an miRNA library and identified miRNAs that regulate CSR. One such miRNA, miR-181b, impairs CSR when expressed in activated B cells, and results in the down-regulation of AID mRNA and protein levels. We found that the AID 3′ untranslated region contains multiple putative binding sequences for miR-181b and that these sequences can be directly targeted by miR-181b. Overall, our results provide evidence for a new regulatory mechanism that restricts AID activity and can therefore be relevant to prevent B cell malignant transformation.

Epstein-Barr Virus-Induced miR-155 Attenuates NF-κB Signaling and Stabilizes Latent Virus Persistence

Abstract: Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that has oncogenic potential, especially among immunocompromised individuals (14, 24). EBV DNA and gene products have been identified in the majority of endemic forms of Burkitt's lymphomas and nasopharyngeal carcinomas, as well as in gastric carcinomas, T-cell lymphomas, Hodgkin's disease Reed-Sternberg cells, and the majority of central nervous system lymphoma found in human immunodeficiency virus-associated AIDS. EBV can infect B lymphocytes and epithelial cells in the oropharynx and establishes long-term latent infection in memory B lymphocytes. Primary infection of B lymphocytes with EBV leads to a germinal-center-like reaction and the continuous proliferation and expansion of the latently infected B cells (32, 38). This continuous proliferation successfully immortalized B lymphocytes if T cells have been eliminated or suppressed by drugs such as cyclosporine. The efficiency of EBV immortalization of primary B cells in vitro has been a useful tool for cell culture and evidence for its oncogenic potential during immune suppression. Immortalization of primary B cells by EBV occurs with relatively high efficiency in the absence of an antiviral T-cell response (38). During the immortalization process, EBV expresses several viral genes that have known growth-transforming activity. The EBNA2 mimics intracellular Notch by activating CBF1 (also known as CSL/RBPjk)-bound genes, while EBNA3C disrupts cell cycle checkpoints. Latency membrane protein 1 (LMP1) and LMP2 mimic constitutively active CD40 coreceptor and B-cell receptor, respectively. LMP1 binds members of the TRAF family through its intracellular domain to activate the NF-κB pathway (13, 29). However, the EBV activation and signaling mechanisms are complex and distinct from the endogenous receptors, and many of the details of gene reprogramming necessary for B-cell immortalization remain undefined. MicroRNAs (miRNAs) are an abundant class of noncoding small RNAs ∼21 nucleotides that can affect gene expression by posttranscriptional regulation of mRNA (6, 11). miRNAs typically base pair with sequences in the 3′ untranslated region (UTR) of multiple mRNAs to inhibit mRNA translation or promote their degradation. miRNAs have been shown to play important roles in various cellular and pathogenic processes, including cellular development, immunological response, and carcinogenesis (2, 7, 39). Viruses, including EBV and Kaposi's sarcoma-associated herpesvirus (KSHV), encode multiple miRNAs, referred to as viral miRNAs, which may mimic cellular miRNAs and modulate cellular and viral gene expression (4, 9, 23). Virus infection can also affect cellular miRNA expression (20, 36). Among the known miRNAs, miR-155 has been strongly implicated in the normal and malignant cell development of B lymphocytes. miR-155 is derived from the B-cell integration cluster (BIC)-associated noncoding RNA precursor (30). BIC and miR-155 were found to be overexpressed in a variety of B-cell malignancies, including Hodgkin's lymphomas, diffuse large cell lymphoma (16), and EBV-positive tumors, including those found in posttransplant lymphoproliferative disease (15, 36). Transgenic mice expressing high levels of miR-155 demonstrate pre-B-cell proliferation and develop lymphoblastic leukemia and high-grade lymphomas (3). miR-155 is induced by inflammatory cytokine response and by B-cell receptor activation (17, 21, 33, 37). miR-155 has been implicated in the normal B-cell developmental processes, including generation of immunoglobulin class-switched plasma cells (34) and germinal center reactions (31). A recent study showed that that miR-155 target seed sequences exists in the 3′ UTR of IKKɛ (8). IKKɛ, the gene product of IKBKE, is a multifunctional IKB kinase protein. IKKɛ had been shown to phosphorylate IRF-3 and IRF-7 to activate interferon (IFN) response to viral infection (27). Recent studies also showed that IKKɛ regulated NF-κB activation directly by phosphorylating the C-terminal domain of RelA and c-Rel through which IKKɛ exerts its oncogenic function (1, 12). In the present study, we present evidence that EBV immortalization of B lymphocytes correlates with induction of miR-155. We show that miR-155 attenuates NF-κB signaling in EBV-positive B lymphocytes and that miR-155 is important for the stable maintenance of EBV genomes during latent infection.