miR-21 Gene Research Articles

High-Resolution Comparative Genomic Hybridization of Mirna Genes In Therapy-Related AML Identifies a Somatic Deletion of MiR-223

AbstractAbstract 2759MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and diverse cellular processes. Expression profiling of miRNAs has identified dysregulated miRNAs in many cancers, including acute myeloid leukemia (AML). However, the mechanism for altered miRNA gene expression and the frequency of miRNA gene mutations in AML is largely unknown. We performed next-generation sequencing and high-resolution comparative genomic hybridization (CGH) to determine the frequency of miRNA gene mutations in 30 patients with therapy-related AML (t-AML). The sequencing is completed, and final analysis is underway. Herein, we report the results of the CGH. We designed custom CGH arrays for all 835 miRNA genes in miRBase (version 14) and 44 genes involved in miRNA processing. Average probe spacing was 30 bp for miRNA genes and 80 bp for miRNA processing genes and 10 kb flanking regions were interrogated for all genes. In each case, genomic DNA from leukemic blasts was compared with DNA from a skin biopsy from that patient. The median age of the 30 patients with t-AML was 49.2 years (25-77) with M:F ratio 13:17. The mean blast % was 81% (31-100). Consistent with previous reports, many of these patients had an abnormal karyotype with abnormalities of chromosome 5 and 7, 13% and 17% respectively. As expected, CGH analysis confirmed copy number alterations already identified by cytogenetics. In addition, we identified a single t-AML sample (from a male patient) that carried a small (435 kb) hemizygous deletion of miR-223 on chromosome × that was not apparent by cytogenetics. Quantitative PCR of genomic DNA confirmed the loss of the miR-223 gene, and real time RT-PCR demonstrated loss of miR-223 expression. Of note, miR-223 has been implicated in the regulation of granulopoiesis, and mice lacking miR-223 display a myeloproliferative phenotype (Johnnidis, Nature 2008). We screened an additional 27 AML patients for miR-223 expression and identified 3 other samples with miR-223 expression 2 standard deviations below the normal (based on CD34+ cells from healthy donors). No copy number alterations in miR-223 were detected in these patients, suggesting epigenetic silencing of miR-223. Consistent with this possibility, one of these patients carried a t(8;21), which has been shown to epigenetically silence miR-223 (Fazi, Cancer Cell 2007). The mechanism by which miR-223 is silenced in the other two AML samples is under investigation. In summary, cytogenetically silent deletions of miRNA genes are uncommon in t-AML. Loss of miR-223 expression can occur through somatic mutation or epigenetic silencing and is likely to contribute to leukemic transformation in a subset of patients with AML. Disclosures:No relevant conflicts of interest to declare.

NF-κB targets miR-16 and miR-21 in gastric cancer: involvement of prostaglandin E receptors

Cigarette smoke is one of the risk factors for gastric cancer and nicotine has been reported to promote tumor growth. Deregulation of microRNA (miRNA) and cyclooxygenase-2 (COX-2) expressions are hallmarks of many cancers including gastric cancer. Here, we used an miRNA array platform covering a panel of 95 human miRNAs to examine the expression profile in nicotine-treated gastric cancer cells. We found that miR-16 and miR-21 were upregulated upon nicotine stimulation, transfection with anti-miR-16 or anti-miR-21 significantly abrogated cell proliferation. In contrast, ectopic miR-16 or miR-21 expression exhibited a similar stimulatory effect on cell proliferation as nicotine. Nicotine-mediated IkappaBα degradation and nuclear factor-kappa B (NF-κB) translocation dose-dependently. Knockdown of NF-κB by short interfering RNA (siRNA) or specific inhibitor (Bay-11-7085) markedly suppressed nicotine-induced cell proliferation and upregulation of miR-16 and miR-21. Interestingly, NF-κB-binding sites were located in both miR-16 and miR-21 gene transcriptional elements and we showed that nicotine enhanced the binding of NF-κB to the promoters of miR-16 and miR-21. Furthermore, activation of COX-2/prostaglandin E₂ (PGE₂) signaling in response to nicotine was mediated by the action of prostaglandin E receptors (EP2 and EP4). EP2 or EP4 siRNA or antagonists impaired the nicotine-mediated NF-κB activity, upregulation of miR-16 and miR-21 and cell proliferation. Taken together, these results suggest that miR-16 and miR-21 are directly regulated by the transcription factor NF-κB and yet nicotine-promoted cell proliferation is mediated via EP2/4 receptors. Perhaps this study may shed light on the development of anticancer drugs to improve the chemosensitivity in smokers.

Inflammatory cytokines regulate microRNA-155 expression in human retinal pigment epithelial cells by activating JAK/STAT pathway

Inflammatory response of the retinal pigment epithelium plays a critical role in the pathogenesis of retinal degenerative diseases such as age-related macular degeneration. Our previous studies have shown that human retinal pigment epithelial (HRPE) cells, established from adult donor eyes, respond to inflammatory cytokines by enhancing the expression of a number of cytokines and chemokines. To investigate the role of microRNA (miRNA) in regulating this response, we performed microarray analysis of miRNA expression in HRPE cells exposed to inflammatory cytokine mix (IFN-γ + TNF-α + IL-1β). Microarray analysis revealed ∼11-fold increase in miR-155 expression, which was validated by real-time PCR analysis. The miR-155 expression was enhanced when the cells were treated individually with IFN-γ, TNF-α or IL-1β, but combinations of the cytokines exaggerated the effect. The increase in miR-155 expression by the inflammatory cytokines was associated with an increase in STAT1 activation as well as an increase in protein binding to putative STAT1 binding elements present in the MIR155 gene promoter region. All these activities were effectively blocked by JAK inhibitor 1. Our results show that the inflammatory cytokines increase miR-155 expression in human retinal pigment epithelial cells by activating the JAK/STAT signaling pathway.

Abstract 3001: MicroRNAs regulating wnt signaling are downregulated in malignant pleural mesothelioma

Abstract Malignant pleural mesothelioma is a rapidly fatal disease whose diagnosis, particularly through less invasive techniques such as analysis of pleural effusion, can be challenging. Currently, a commercially available diagnostic test based on microRNA expression patterns is purported to distinguish between mesothelioma and lung adenocarcinoma. Yet, the biological basis of this technology has not been reported in the literature, and little research has been aimed at determining how differential miRNA expression contributes to the differences in pathogenesis between these diseases, both of which can be caused by asbestos exposure. We sought to illuminate the molecular differences between mesothelioma and lung adenocarcinoma by using microRNA microarrays to identify patterns in the most differentially expressed microRNAs. From this, we identified a panel of microRNAs, including members of the miR-200 gene family, that were all downregulated in malignant pleural mesothelioma compared to lung adenocarcinoma. Using the more sensitive detection method of quantitative RT-PCR on an independent series of tumors, we validated the specificity of these alterations in 100 malignant pleural mesotheliomas and 32 lung adenocarcinomas. Further examination of the relationship between these microRNAs revealed that they are predicted to act as redundant regulators of wnt signaling, and suggests a role for this pathway in tumor progression. This research points to novel approaches utilizing miRNAs whose decreased expression is unique to mesothelioma as potentially suitable for rapid diagnosis, and reveals important new targets for the treatment of this deadly disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3001.

A miR-21 hairpin structure-based gene knockdown vector

RNA interference (RNAi) is widely used to study gene functions as a reverse genetic means from first-generation siRNA to second-generation short hairpin RNA (shRNA) or the newly developed microRNA (shRNA-miR). Here we report a gene knockdown vector system based on the mouse miR-21 hairpin structure. In this system, the pre-miRNA hairpin of the miR-21 gene was modified by replacing the 22-nucleotide mature sequence with shRNA sequences that target genes of interest, flanked by 160-bp upstream and 65-bp downstream sequences of the mouse pre-miR-21. We tested this system by knocking down the enhanced green fluorescence protein (EGFP) reporter gene using different vectors, in which shRNA-miR was driven by the polymerase II (pol II) promoter. We found that miR-21 hairpin-based shRNA-miR can be directly placed under pol II promoter, like UbC or CMV promoter to knockdown the gene of interest. To facilitate the wide application of the miR-21 hairpin-based gene knockdown system, we further knocked down the endogenous gene lamin (A/C), which showed that endogenous lamin A/C expression can be efficiently silenced using the miR-21 hairpin-based lentiviral vector. The miR-21 hairpin-based gene knockdown vector will provide a new genetic tool for gene functional studies in vitro and in vivo.

Cell-cycle regulator E2F1 and microRNA-223 comprise an autoregulatory negative feedback loop in acute myeloid leukemia

AbstractTranscription factor CCAAT enhancer binding protein α (C/EBPα) is essential for granulopoiesis and its function is deregulated in leukemia. Inhibition of E2F1, the master regulator of cell-cycle progression, by C/EBPα is pivotal for granulopoiesis. Recent studies show microRNA-223 (miR-223), a transcriptional target of C/EBPα, as a critical player during granulopoiesis. In this report, we demonstrate that during granulopoiesis microRNA-223 targets E2F1. E2F1 protein was up-regulated in miR-223 null mice. We show that miR-223 blocks cell-cycle progression in myeloid cells. miR-223 is down-regulated in different subtypes of acute myeloid leukemia (AML). We further show that E2F1 binds to the miR-223 promoter in AML blast cells and inhibits miR-223 transcription, suggesting that E2F1 is a transcriptional repressor of the miR-223 gene in AML. Our study supports a molecular network involving miR-223, C/EBPα, and E2F1 as major components of the granulocyte differentiation program, which is deregulated in AML.

Downregulated microRNAs in the differential diagnosis of malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is a rapidly fatal disease whose diagnosis, particularly through less invasive techniques such as analysis of pleural effusion, can be challenging. Currently, a commercially available diagnostic test based on microRNA (miRNA) expression patterns is purported to distinguish between mesothelioma and lung adenocarcinoma. Yet, the biological basis of this technology has not been reported in the literature, and little research has been aimed at determining how differential miRNA expression contributes to the differences in pathogenesis between these diseases, both of which can be caused by asbestos exposure. We sought to illuminate the molecular differences between mesothelioma and lung adenocarcinoma by using miRNA microarrays to identify patterns in the most differentially expressed miRNAs. From this, we identified a panel of miRNAs, including members of the miR-200 gene family, that were all downregulated in MPM compared to lung adenocarcinoma. Using the more sensitive detection method of quantitative RT-PCR on an independent series of tumors, we validated the specificity of these alterations in 100 MPMs and 32 lung adenocarcinomas. Statistical analysis reveals that these miRNAs exceed the current recommendations for biomarkers and could greatly aid in the differential diagnosis. Further examination led us to predict that they act as redundant regulators of wnt signaling and suggests a role for this pathway in tumor progression. This research points to novel approaches using miRNAs whose decreased expression is unique to mesothelioma as potentially suitable for rapid diagnosis and reveals prospective new targets for the treatment of this deadly disease.

The Modulation of MicroRNAs by Type I IFN through the Activation of Signal Transducers and Activators of Transcription 3 in Human Glioma

Type I IFNs are involved in double-stranded RNA responses. Here, we investigated the possibility that IFN-beta may induce or downregulate cellular microRNAs (miRNA) in human neoplasms and thereby use the RNA interference system to show antitumor effects. Because of its known connection to glioma biology, we focused on miR-21 among seven miRNAs influenced by IFN-beta. We analyzed the effect of IFN-beta treatment on miR-21 expression in glioma cells and intracranial glioma xenografts. IFN-beta treatment reduced miR-21 expression in glioma cells markedly, and IFN-beta administration suppressed the growth of glioma-initiating cell-derived intracranial tumors. The levels of primary miR-21 gene transcripts, precursor miR-21, and mature miR-21 decreased 6 hours after the addition of IFN-beta, indicating that the reduction in miR-21 levels was due to transcriptional suppression. We did reporter assays to elucidate the IFN-beta-mediated suppression of miR-21; the addition of signal transducers and activators of transcription 3 (STAT3)-expressing vectors induced the IFN-beta-mediated suppression of miR-21, whereas STAT3-inhibiting agents inhibited the miR-21 suppression. Thus, the results of our study show that the downregulation of miR-21 contributes to the antitumor effects of IFN-beta and that miR-21 expression is negatively regulated by STAT3 activation. These results highlight the importance of understanding the transcriptional regulation of the miRNAs involved in oncogenesis.

MicroRNA Mir-155 and Myb Proto-Oncogene Family Members Cooperate in Pathogenesis of Chronic Lymphocytic Leukemia.

Abstract 58 Introduction:Role of small non-coding microRNAs (miRNA) in hematopoiesis has been recently established by studies demonstrating increased levels of miR-155 in chronic lymphocytic leukemia (CLL) (Fulci 2007, Marton 2008). PU.1 is an ETS family transcription factor controling myelo-lymphoid differentiation and is directly negatively regulated by miR-155 (Vigorito 2007). Our aim of this study is to determine mechanisms of miR-155 upregulation in CLL pathogenesis and the role of PU.1 downregulation in the process. Methods: miRNA and mRNA levels were determined by qPCR and Affymetrix mRNA expression profiling in peripheral blood mononuclear cells (PBMC) and in purified B cells. Control (N=13) and CLL patients (N=66) were studied. All patients were subgrouped according to cytogenetics (FISH) and Rai status. Protein-DNA localization assays were done using chromatin immunoprecipitation. Results: miR-155 is significantly upregulated in both CLL patient PBMCs and a subset of sorted B-cells whereas PU.1 and its target genes are repressed in all CLL subgroups. Indeed, expression profiling analysis of CLL samples identified a broad repression of ∼80 miR-155 targets (among them key transcriptional regulators FOS, SATB1, MEF2A, MYBL1, SIRT1, MECP2 and CEBPB) and ∼380 repressed PU.1 target genes, among them regulators of hematopoietic homeostasis (FOS, CSF1R, CSF2R, IL4R, IL21R) and apoptosis (BID, BIRC3). Next, we have studied the mechanism of miR-155 gene (also known as BIC) upregulation in CLL. Wehave newly identified a regulatory CpG island (BIC-CpG) upstream of miR-155 BIC gene that contains DNA binding motifs for E-box transcription factors and is not mutated in CLL patients. Two E-box-binding proteins, MYB and MYBL2, are significantly upregulated in CLL patient PBMCs as well as in a subset of sorted B-cells in all CLL subgroups. Furthermore in primary CLL cells, MYB protein presence is significantly enriched at BIC-CpG alongside a marked enrichment with transcriptionally active chromatin mark histone H3K9Acetyl. To further study the role of MYB in transactivation of the BIC-CpG we have prepared reporter constructs and found that MYB indeed activates BIC-CpG and downstream transcription. Apart from miR-155/BIC, expression profiling analysis identified additional ∼50 upregulated MYB targets, among them cancer-related genes such as CA1, MCM4, BCL2, PDCD4, and CXCR4. Functional assays using siRNA inhibition of PU.1 in normal PBMCs result in upregulation of miR-155 and MYB, indicating that silencing of PU.1 and upregulation of MYB and its target miR-155 may represent an important mechanism of CLL pathogenesis. Conclusion: Our data propose a mechanistic relationship between PU.1 and its negative regulator miR-155 in CLL. Our data also demonstrate that miR-155 is transcriptionally activated by MYB family of E-box binding proteins. Manipulation of these mechanistic relationships may harbor a potential for molecular therapies against CLL. (Grants NR9021-4, 10310-3, 2B06077) Disclosures:No relevant conflicts of interest to declare.

The Interplay Between Microrna-223 and E2F1 Regulates Cell Cycle Control During Granulopoiesis.

Abstract 255Transcription factor CCAAT enhancer binding protein α (C/EBPα) functions as a master regulator of granulocyte development by co-ordinating cell cycle inhibition and differentiation. Recent findings demonstrate that deregulation of C/EBPα is a critical step in the development of acute myeloid leukemia (AML). Inhibition of E2F1, the key regulator of cell cycle progression by C/EBPα is essential for granulopoiesis and disruption of this function of C/EBPα leads to leukemia. The mechanism with which C/EBPα inhibits E2F1 in granulopoiesis is poorly understood. Recent advances in our understanding about microRNAs suggest that these molecules have profound impact in gene expression programmes. Also, deregulation of microRNAs has been shown as a hall mark of many cancers including leukemia. microRNA-223 (miR-223) is upregulated by C/EBPα during granulopoiesis. The pivotal role of miR-223 in granulopoiesis is shown by the finding that mice deficient for miR-223 display defects in granulopoiesis. In this study, we explored the role of miR-223 in the cell cycle inhibition function of C/EBPα. Computational analysis by using programmes such as Target Scan suggests that E2F1 is a putative target of miR-223. Luciferase assays using 3'UTR of E2F1 suggest E2F1 is a potential target of miR-223. Western blot analysis using bone marrow cells isolated from miR-223 null mice shows accumulation of E2F1 protein levels. Interestingly, E2F1 protein levels were downregulated during miR-223 overexpression in myeloid cells. Analysis of miR-223 by quantitative Real-Time RT-PCR in AML patient samples shows that miR-223 is downregulated in different subtypes of AML. Proliferation assays, cell cycle analysis and BrdU assays show that miR-223 functions as an inhibitor of myeloid cell cycle progression. Several studies have reported the ability of E2F1 to block granulocytic differentiation. We next analysed whether E2F1 is inhibiting myeloid differentiation through miR-223. Promoter assays show that E2F1 inhibits the miR-223 promoter activity. By using Chromatin immunoprecipitation assays, we found that E2F1 binds to miR-223 promoter in leukemia derived cell lines and this binding is reversed during granulocytic differentiation. We also observed that E2F1 is bound to the miR-223 promoter in blast cells isolated from AML patients as analysed by chromatin immunoprecipitation assays. In addition, we show that overexpression of E2F1 leads to down regulation of miR-223 levels in myeloid cells. All these data suggest that E2F1 functions as a transcriptional repressor of the miR-223 gene. Taken together, our data suggest that granulopoiesis is regulated by the interplay between miR-223 and E2F1 and deregulation of this interplay may lead to the development of AML. Overexpression of miR-223 could be a potential strategy in the treatment of AML patients in which E2F1 inhibition by C/EBPα is deregulated. Disclosures:No relevant conflicts of interest to declare.

Locked Nucleic Acid In situ Hybridization Analysis of miR-21 Expression during Colorectal Cancer Development

To better understand microRNA miR-21 function in carcinogenesis, we analyzed miR-21 expression patterns in different stages of colorectal cancer development using in situ hybridization (ISH). Locked nucleic acid (LNA)/DNA probes and a biotin-free tyramide signal amplification system were used in ISH analyses of miRNA expression. Conditions for specific detection of miR-21 were determined using human cell lines and miR-21-expressing lentiviral vectors. Expression was determined in 39 surgically excised colorectal tumors and 34 endoscopically resected colorectal polyps. In the surgical samples, miR-21 expression was much higher in colorectal cancers than in normal mucosa. Strong miR-21 expression was also observed in cancer-associated stromal fibroblasts, suggesting miR-21 induction by cancer-secreted cytokines. Protein expression of PDCD4, a miR-21 target, was inversely correlated with miR-21 expression, confirming that miR-21 is indeed a negative regulator of PDCD4 in vivo. In the endoscopic samples, miR-21 expression was very high in malignant adenocarcinomas but was not elevated in nontumorigenic polyps. Precancerous adenomas also frequently showed miR-21 up-regulation. Using the LNA-ISH system for miRNA detection, miR-21 was detectable in precancerous adenomas. The frequency and extent of miR-21 expression increased during the transition from precancerous colorectal adenoma to advanced carcinoma. Expression patterns of miR-21 RNA and its target, tumor suppressor protein PDCD4, were mutually exclusive. This pattern may have clinical application as a biomarker for colorectal cancer development and might be emphasized by self-reinforcing regulatory systems integrated with the miR-21 gene, which has been previously shown in cell culture.

The miR-200 Family: Central Player for Gain and Loss of the Epithelial Phenotype

The miR-200 Family: Central Player for Gain and Loss of the Epithelial Phenotype

Estradiol downregulates miR-21 expression and miR-21 gene targets in MCF-7 breast cancer cells.

Abstract Abstract #3051 Select changes in microRNA (miRNA) expression correlate with diagnostic markers used in breast cancer therapies, e.g., estrogen receptor α (ERα) and tumor grade. However, no one has examined how estradiol (E2) regulates miRNA expression in breast cancer cells. Here miRNA microarrays were used to identify miRNA genes regulated by E2 in MCF-7 breast cancer cells. Twenty-five miRNAs were E2-regulated and select results were confirmed by quantitative-PCR. E2 reduced the expression of miR-21, an oncogene, and repression was inhibited by the antiestrogen ICI 182,780, ERα knockdown by siRNA, and by pre-treatment with Actinomycin D or cycloheximide. Thus, E2-ERα reduces miR-21 expression by primary transcriptional and secondary target activities. The effect of E2 on luciferase reporters containing the miRNA recognition elements (MREs) from the 3'-UTRs of 8 putative miR-21 regulated genes: CCM1, SKI, TGFB1, PDCD4, RAB6A, RAB6C, RASA1, and RASGRP1 was tested in MCF-7 cells. E2 increased luciferase with the MREs from TGFB1, PDCD4, RASA1, and RASGRP1, and from the full length 3' UTR of the PDCD4 gene, consistent with an E2-mediated decrease in miR-21 resulting in increased stability of Renilla mRNA and increased luciferase activity. Antisense to miR-21 increased luciferase from the TGFB1,PDCD4, RAB6A, and RASGRP1 reporters, consistent with these MREs being bona fide miR-21 targets. The E2-mediated decrease in miR-21 correlated with increased expression of miR-21-targets PDCD4, PTEN, and Bcl-2 at the protein level and these increases were blocked by AS to miR-21 and by siRNA knockdown of ERα. These results identify miR-21 as an ERα- regulated miRNA in MCF-7 cells. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3051.

The Mir-17-19b Microrna Cluster Quantitatively Regulates the Self- Renewing Leukemia Stem Cell Compartment in MLL Leukemia

The critical molecular determinants for leukemia stem cell (LSC) generation and maintenance are largely unknown. While the majority of studies focus on investigating key transcription factors and cytokines involved in hematological malignancies, very little is known about the roles of recently discovered microRNAs in leukemogenesis. Bioinformatic analysis of the Mouse Retroviral Tagged Cancer Gene Database (Neal Copeland et al.) revealed the 5′ regulatory region of the miR-142 gene as a recurrent target for retroviral integrations in a variety of mouse leukemia and lymphoma cancer models, including MLL-induced leukemia, thereby implicating miR-142 as a potential collaborating oncomir in hematologic malignancies. Subsequent expression analyses of differentially expressed microRNAs in LSC-enriched cell fractions (c-kit+) from MLL leukemias in comparison with various stages of normal hematopoietic progenitors indicated that the miR-17-5p family of microRNAs is highly expressed in LSC-enriched fractions and their possible normal counterparts, granulocyte-macrophage progenitors. Moreover, the expression of miR-142 and miR-17-5p family microRNAs was substantially reduced upon differentiation and loss of self-renewal in cells transformed by the conditional MLL-ENL-ER oncogene following withdrawal of tamoxifen. Forced expression of the miR-17-19b polycistron or miR-142 substantially shortened the latency for MLL leukemia development. In particular, leukemias expressing high levels of the miR-17-19b polycistron displayed a higher frequency of LSCs due to an efficient block of differentiation and enhanced proliferation associated with reduced expression of p21, a cyclin dependent kinase inhibitor previously implicated as a direct target of miR-17-19b microRNAs. Knock down of p21 in MLL oncogene transduced cells recapitulates the MLL-leukemic phenotype induced by over-expression of the miR-17-19b cluster including a significant decrease in MLL leukemia latency. This observation validates p21 as a biologically relevant in vivo target of the miR-17-19b polycistron in MLL leukemia. Thus, microRNAs are pathogenically important for quantitatively modulating the self-renewing LSC compartment in a murine model of MLL leukemia. Potential requirements of the miR-17-19b microRNA cluster for induction and/or maintenance of MLL leukemia are currently under study.

A Molecular Network Comprising MicroRNA-223, E2F1 and C/Ebpα in Granulopoiesis and in Acute Myeloid Leukemia.

MicroRNAs play crucial roles in gene expression programmes and have been demonstrated to have major influence in various biological processes. Recent findings suggest aberrant regulation of microRNAs is a hall mark of many cancers including leukemia. MicroRNA-223 (miR-223) is regulated by the transcription factor CCAAT enhancer binding protein α (C/EBPα) and is upregulated during granulopoiesis. miR-223 mutant mice display defects in granulopoiesis pointing out the importance of miR-223 during granulopoiesis. Recent studies suggest that loss of function or expression of C/ EBPα is a major step in the development of acute myeloid leukemia (AML). Using an inducible cell line model, we show that C/EBPα upregulates microRNA-223 expression during granulopoiesis. Based on these findings, we hypothesized that miR-223 could be downregulated in human AML. Here we report that miR-223 is downregulated in different subtypes of AML as analysed by quantitative Real-Time RT-PCR. We investigated what are the critical targets of miR-223 during granulopoiesis. Computational analysis suggests that E2F1, the transcription factor that promotes cell cycle progression which is inhibited by C/EBPα during granulopoiesis, could be a putative target of miR-223. By luciferase assay using 3'UTR of E2F1, we show that E2F1 is a potential target of miR-223. miR-223 downregulates E2F1 by translational repression as revealed by reduction in E2F1 protein level. Silencing of miR-223 leads to upregulation of E2F1 protein level as analyzed by Western blot analysis. Proliferation assays as well as cell cycle analysis demonstrate that miR-223 blocks cell cycle progression in myeloid cells. Interestingly, sequence analysis of miR-223 promoter revealed putative E2F1 binding sites. We demonstrate that E2F1 inhibits the microRNA-223 promoter activity through its transactivation domain as shown by promoter assay. Furthermore, overexpression of E2F1 down regulates the expression of miR-223, suggesting E2F1 acting as a transcriptional repressor of the miR-223 gene. Meanwhile, C/EBPα transactivates miR-223 promoter activity. We also report that E2F1 is able to block granulocytic differentiation. Recent studies demonstrate that disruption of E2F1 inhibition by C/EBPα leads to leukemia, pointing out the significance of E2F1 inhibition in the development of AML. Our data support a circuitry comprising miR-223, C/EBPα and E2F1 as major components of the granulocyte differentiation programme, which is deregulated in AML. Manipulation of miR-223 could be therapeutically relevant in AML subtypes in which E2F1 inhibition is deregulated.

Small‐Molecule Inhibitors of MicroRNA miR‐21 Function

MicroRNAs (miRNAs) have recently emerged as an important class of gene regulators, and their misregulation has been linked to a variety of cancers. Small molecule inhibitors of miRNAs would be important tools to elucidate the detailed mechanisms of miRNA function and provide lead structures for the development of new therapeutics. We are reporting a cellular screen for miRNA pathway inhibitors and the first small molecule modifiers of miRNA function.

‘microRNA’ Review Series The ongoing microRNA revolution and its impact in biology and medicine

‘microRNA’ Review Series First identified more than a decade ago in C. elegans, microRNAs have emerged as key molecular players in virtually all the cellular processes studied to date [1]. The scientific community has witnessed an arguably unparalleled expansion in the number of publications focusing on microRNA biology, from only a handful in 2001 to well over a thousand during the course of last year. In the meantime, the number of experimentally confirmed microRNAs has increased, now exceeding 500 in most studied organisms, including mammals. Many microRNAs exhibit a remarkable degree of evolutionary conservation between birds, rodents and primates, a reflection of their critical importance for fundamental cellular processes. Novel bioinformatic approaches and increasingly advanced cloning technologies could lead to a further enrichment of the microRNAs world in the years to come. Mechanistically, microRNAs interfere with gene expression at post-transcriptional level, subsequently to base pairing to partially complementary sites within the 3'UTR of the targets [2]. The early prevailing paradigm divided microRNAs actions along evolutionary lines. Thus, in plants they were thought to interfere with messenger RNA stability, similar to siRNAs, whereas in animal cells inhibition of protein translation was proposed to be their dominant, if not the exclusive, action. More recently, however, this dichotomy has been challenged by experimental data, as mRNA degradation seems relatively common in mammalian cells [3]. Another amendment to the translation inhibition paradigm was issued last year, with the identification of specific microRNAs that enhance rather than inhibit translation of specific protein. For example, miR-369–3 was shown to stimulate the translation of tumour necrosis factor-α during cell cycle arrest, contrary to its effect in proliferating cells [4]. It is entirely possible that this case represents only the tip of the iceberg, and under specific circumstances a multitude of microRNAs could switch between inhibition and stimulation of protein translation. In parallel to the rapid gain in biological knowledge, the commercially available technology has matured, as multiple life-science companies (such as LC sciences; Houston, TX, USA, Ambion, Inc (now part of Applied Biosystems); Austin, TX, USA, Exiqon; Vedback, Denmark, Invitrogen; Carlsbad, CA, USA) now provide a wide range of reagents and microarray platforms. Profiling approaches have started to shed light into the dynamic of the ‘microtranscriptome’ in diverse processes, such as development, metabolic processes or signalling. Additionally, signatures for a wide variety of diseases have been generated, leading the way to more focused investigations into the roles of specific microRNAs. Arguably the most extensive effort has been undertaken in cancer, one important outcome being microRNA signature(s) attached to virtually every major tumour type [5]. Similar to the case of ‘classic’ genes, oncogenic and tumour-suppressing microRNAs have been identified, which directly regulate apoptosis and/or cell cycle, and in some cases undergo mutations in specific malignancies. Additionally, some cancer-relevant microRNAs have been shown to respond to cellular stresses such as hypoxia, and are thought to play critical roles in cellular adaptation to these [6, 7]. Mechanistic studies of microRNAs have met challenges that are not encountered during the functional dissection of translated genes. This stems from the relative promiscuity of microRNA-dependent gene targeting, characterized by tens or even hundreds of predicted targets, with only partial overlap between the various prediction programs (PicTar, TargetScan, Miranda, etc.). A major step forward has been constituted by the development of animal models that open the way for in vivo investigation of microRNA roles in physiology and pathology. For example, the first transgenic mouse overexpressing miR-155 in B-lymphocytes turned out to be a powerful model of haematological malignancies that naturally overexpress this microRNA (Hodgkin lymphomas, B cell and Burkitt lymphomas) [8]. Conversely, mir-155 gene knockouts developed a wide dysfunction of the immune response, involving T, B and dendritic cells [9]. The involvement of microRNAs in disorders that sit at the top of morbidity and mortality lists (cancer, inflammation, and possibly, cardiovascular pathology and stroke [10]) can be viewed as an opportunity for novel therapeutic approaches. Along these lines, progress has been achieved for the targeting of select disease-relevant microRNAs, with several drug candidates making promising strides in clinical trials [11]. The plethora of exciting developments in the microRNA field, from basic science to translational applications, serve as fertile background for the review series hosted by the Journal of Cellular and Molecular Medicine. It is also an opportunity to wholeheartedly invite researchers from the field and beyond to contribute to the journal with novel angles to this fast-evolving area.

Hypoxia induces microRNA miR-210 in vitro and in vivo: Ephrin-A3 and neuronal pentraxin 1 are potentially regulated by miR-210

Shortage of oxygen is one of the prime stress conditions in tissues. In this study, we looked for microRNAs expressed during hypoxia and showed that miR-210 expression was upregulated in response to hypoxia in vitro and in vivo. An active form of the HIF-1α induced the expression of miR-210, showing the involvement of the HIF-1 signaling pathway in miR-210 gene transcription. Furthermore, miR-210 was shown to bind to the predicted target sites of ephrin-A3 or neuronal pentraxin 1, causing repression in luciferase reporter activity. Contrary to the microRNA-mediated repression hypothesis, ephrin-A3 was expressed at very high levels in post-ischemic mouse hippocampus in vivo. Thus, the regulatory effects of miR-210 on its targets in vivo need to be further characterized.

MiR-21 Gene Expression Triggered by AP-1 Is Sustained through a Double-Negative Feedback Mechanism

miR-21 has been reported to be highly expressed in various cancers and to be inducible in a human promyelocytic cell line, HL-60, after phorbol 12-myristate 13-acetate (PMA) treatment. To examine molecular mechanisms involved in miR-21 expression, we analyzed the structure of the miR-21 gene by determining its promoter and primary transcripts. We show that activation protein 1 (AP-1) activates the miR-21 transcription in conjugation with the SWI/SNF complex, after PMA stimulation, through the conserved AP-1 and PU.1 binding sites in the promoter identified here. The previous findings of enhanced miR-21 expression in several cancers may therefore reflect the elevated AP-1 activity in these carcinomas. A single precursor RNA containing miR-21 was transcribed just downstream from the TATA box in this promoter, which is located in an intron of a coding gene, TMEM49. More important, expression of this overlapping gene is completely PMA-independent and all its transcripts are polyadenylated before reaching the miR-21 hairpin embedding region, indicating that miRNAs could have their own promoter even if overlapped with other genes. By available algorithms that predict miRNA target using a conservation of sequence complementary to the miRNA seed sequence, we next predicted and confirmed that the NFIB mRNA is a target of miR-21. NFIB protein usually binds the miR-21 promoter in HL-60 cells as a negative regulator and is swept off from the miR-21 promoter during PMA-induced macrophage differentiation of HL-60. The translational repression of NFIB mRNA by miR-21 accelerates clearance of NFIB in parallel with the simultaneous miR-21-independent transcriptional repression of NFIB after PMA stimulation. Since exogenous miR-21 expression moderately induced endogenous miR-21, an evolutionarily conserved double-negative feedback regulation would be operating as a mechanism to sustain miR-21 expression.

MiR-210 links hypoxia with cell cycle regulation and is deleted in human epithelial ovarian cancer

Tumor growth results in hypoxia. Understanding the mechanisms of gene expression reprogramming under hypoxia may provide important clues to cancer pathogenesis. We studied miRNA genes that are regulated by hypoxia in ovarian cancer cell lines by TaqMan miRNA assay containing 157 mature miRNAs. MiR-210 was the most prominent miRNA consistently stimulated under hypoxic conditions. We provide evidence for the involvement of the HIF signaling pathway in miR-210 regulation. Biocomputational analysis and in vitro assays demonstrated that e2f transcription factor 3 (e2f3), a key protein in cell cycle, is regulated by miR-210. E2F3 was further confirmed to be downregulated at the protein level upon induction of miR-210. Importantly, we found remarkably high frequency of miR-210 gene copy deletions in ovarian cancer patients (64%, n=114) and that gene copy number correlates with miR-210 expression levels. Taken together, our results indicate that miR-210 plays a crucial role in tumor onset as a key regulator of the hypoxia response and provide evidence for a link between hypoxia and the regulation of cell cycle.