The RNA editing enzyme ADAR2 restricts L1 mobility

Adenosine deaminases acting on RNA (ADARs) are enzymes that convert adenosines to inosines in double-stranded RNAs (RNA editing A-to-I). ADAR1 and ADAR2 were previously reported as HIV-1 proviral factors. The aim of this study was to investigate the composition of the ADAR2 ribonucleoprotein complex during HIV-1 expression. By using a dual-tag affinity purification procedure in cells expressing HIV-1 followed by mass spectrometry analysis, we identified 10 non-ribosomal ADAR2-interacting factors. A significant fraction of these proteins was previously found associated to the Long INterspersed Element 1 (LINE1 or L1) ribonucleoparticles and to regulate the life cycle of L1 retrotransposons. Considering that we previously demonstrated that ADAR1 is an inhibitor of LINE-1 retrotransposon activity, we investigated whether also ADAR2 played a similar function. To reach this goal, we performed specific cell culture retrotransposition assays in cells overexpressing or ablated for ADAR2. These experiments unveil a novel function of ADAR2 as suppressor of L1 retrotransposition. Furthermore, we showed that ADAR2 binds the basal L1 RNP complex. Overall, these data support the role of ADAR2 as regulator of L1 life cycle

NF-kB and c-Jun induce the expression of the oncogenic miR-221 and miR-222 in prostate carcinoma and glioblastoma cells

MicroRNAs (miRNAs) are potent negative regulators of gene expression involved in all aspects of cell biology. They finely modulate virtually all physiological pathways in metazoans, and are deeply implicated in all main pathologies, among which cancer. Mir-221 and miR-222, two closely related miRNAs encoded in cluster from a genomic region on chromosome X, are strongly upregulated in several forms of human tumours. In this work, we report that the ectopic modulation of NF-kB modifies miR-221/222 expression in prostate carcinoma and glioblastoma cell lines, where we had previously shown their oncogenic activity. We identify two separate distal regions upstream of miR-221/222 promoter which are bound by the NF-kB subunit p65 and drive efficient transcription in luciferase reporter assays; consistently, the site-directed mutagenesis disrupting p65 binding sites or the ectopical inhibition of NF-kB activity significantly reduce luciferase activity. In the most distal enhancer region, we also define a binding site for c-Jun, and we show that the binding of this factor cooperates with that of p65, fully accounting for the observed upregulation of miR-221/222. Thus our work uncovers an additional mechanism through which NF-kB and c-Jun, two transcription factors deeply involved in cancer onset and progression, contribute to oncogenesis, by inducing miR-221/222 transcription

Detection of a MicroRNA Signal in an In Vivo Expression Set of mRNAs

Background. microRNAs (miRNAs) are approximately 21 nucleotide non-coding transcripts capable of regulating gene expression. The most widely studied mechanism of regulation involves binding of a miRNA to the target mRNA. As a result, translation of the target mRNA is inhibited and the mRNA may be destabilized. The inhibitory effects of miRNAs have been linked to diverse cellular processes including malignant proliferation, apoptosis, development, differentiation, and metabolic processes. We asked whether endogenous fluctuations in a set of mRNA and miRNA profiles contain correlated changes that are statistically distinguishable from the many other fluctuations in the data set. Methodology/Principal Findings. RNA was extracted from 12 human primary brain tumor biopsies. These samples were used to determine genome-wide mRN

The Negative Feedback-Loop between the Oncomir Mir-24-1 and Menin Modulates the Men1 Tumorigenesis by Mimicking the “Knudson’s Second Hit”

Multiple endocrine neoplasia type 1 (MEN1) syndrome is a rare hereditary cancer disorder characterized by tumors of the parathyroids, of the neuroendocrine cells, of the gastro-entero-pancreatic tract, of the anterior pituitary, and by non-endocrine neoplasms and lesions. MEN1 gene, a tumor suppressor gene, encodes menin protein. Loss of heterozygosity at 11q13 is typical of MEN1 tumors, in agreement with the Knudson’s two-hit hypothesis. In silico analysis with Target Scan, Miranda and Pictar-Vert softwares for the prediction of miRNA targets indicated miR-24-1 as capable to bind to the 3′UTR of MEN1 mRNA. We investigated this possibility by analysis of miR-24-1 expression profiles in parathyroid adenomatous tissues from MEN1 gene mutation carriers, in their sporadic non-MEN1 counterparts, and in normal parathyroid tissue. Interestingly, the MEN1 tumorigenesis seems to be under the control of a “negative feedback loop” between miR-24-1 and menin protein, that mimics the second hit of Knudson’s hypothesis and that could buffer the effect of the stochastic factors that contribute to the onset and progression of this disease. Our data show an alternative way to MEN1 tumorigenesis and, probably, to the “two-hit dogma”. The functional significance of this regulatory mechanism in MEN1 tumorigenesis is also the basis for opening future developments of RNA antagomir(s)-based strategies in the in vivo control of tumorigenesis in MEN1 carriers

A Ten-microRNA Expression Signature Predicts Survival in Glioblastoma

Glioblastoma (GBM) is the most common and aggressive primary brain tumor with very poor patient median survival. To identify a microRNA (miRNA) expression signature that can predict GBM patient survival, we analyzed the miRNA expression data of GBM patients (n = 222) derived from The Cancer Genome Atlas (TCGA) dataset. We divided the patients randomly into training and testing sets with equal number in each group. We identified 10 significant miRNAs using Cox regression analysis on the training set and formulated a risk score based on the expression signature of these miRNAs that segregated the patients into high and low risk groups with significantly different survival times (hazard ratio [HR] = 2.4; 95% CI = 1.4–3.8; p<0.0001). Of these 10 miRNAs, 7 were found to be risky miRNAs and 3 were found to be protective. This signature was independently validated in the testing set (HR = 1.7; 95% CI = 1.1–2.8; p = 0.002). GBM patients with high risk scores had overall poor survival compared to the patients with low risk scores. Overall survival among the entire patient set was 35.0% at 2 years, 21.5% at 3 years, 18.5% at 4 years and 11.8% at 5 years in the low risk group, versus 11.0%, 5.5%, 0.0 and 0.0% respectively in the high risk group (HR = 2.0; 95% CI = 1.4–2.8; p<0.0001). Cox multivariate analysis with patient age as a covariate on the entire patient set identified risk score based on the 10 miRNA expression signature to be an independent predictor of patient survival (HR = 1.120; 95% CI = 1.04–1.20; p = 0.003). Thus we have identified a miRNA expression signature that can predict GBM patient survival. These findings may have implications in the understanding of gliomagenesis, development of targeted therapy and selection of high risk cancer patients for adjuvant therapy

Identification of Novel SNPs in Glioblastoma Using Targeted Resequencing

High-throughput sequencing opens avenues to find genetic variations that may be indicative of an increased risk for certain diseases. Linking these genomic data to other “omics” approaches bears the potential to deepen our understanding of pathogenic processes at the molecular level. To detect novel single nucleotide polymorphisms (SNPs) for glioblastoma multiforme (GBM), we used a combination of specific target selection and next generation sequencing (NGS). We generated a microarray covering the exonic regions of 132 GBM associated genes to enrich target sequences in two GBM tissues and corresponding leukocytes of the patients. Enriched target genes were sequenced with Illumina and the resulting reads were mapped to the human genome. With this approach we identified over 6000 SNPs, including over 1300 SNPs located in the targeted genes. Integrating the genome-wide association study (GWAS) catalog and known disease associated SNPs, we found that several of the detected SNPs were previously associated with smoking behavior, body mass index, breast cancer and high-grade glioma. Particularly, the breast cancer associated allele of rs660118 SNP in the gene SART1 showed a near doubled frequency in glioblastoma patients, as verified in an independent control cohort by Sanger sequencing. In addition, we identified SNPs in 20 of 21 GBM associated antigens providing further evidence that genetic variations are significantly associated with the immunogenicity of antigens

MicroRNA-218 Is Deleted and Downregulated in Lung Squamous Cell Carcinoma

MicroRNAs (miRNAs) are a family of small, non-coding RNA species functioning as negative regulators of multiple target genes including tumour suppressor genes and oncogenes. Many miRNA gene loci are located within cancer-associated genomic regions. To identify potential new amplified oncogenic and/or deleted tumour suppressing miRNAs in lung cancer, we inferred miRNA gene dosage from high dimensional arrayCGH data. From miRBase v9.0 (http://microrna.sanger.ac.uk), 474 human miRNA genes were physically mapped to regions of chromosomal loss or gain identified from a high-resolution genome-wide arrayCGH study of 132 primary non-small cell lung cancers (NSCLCs) (a training set of 60 squamous cell carcinomas and 72 adenocarcinomas). MiRNAs were selected as candidates if their immediately flanking probes or host gene were deleted or amplified in at least 25% of primary tumours using both Analysis of Copy Errors algorithm and fold change (≥±1.2) analyses. Using these criteria, 97 miRNAs mapped to regions of aberrant copy number. Analysis of three independent published lung cancer arrayCGH datasets confirmed that 22 of these miRNA loci showed directionally concordant copy number variation. MiR-218, encoded on 4p15.31 and 5q35.1 within two host genes (SLIT2 and SLIT3), in a region of copy number loss, was selected as a priority candidate for follow-up as it is reported as underexpressed in lung cancer. We confirmed decreased expression of mature miR-218 and its host genes by qRT-PCR in 39 NSCLCs relative to normal lung tissue. This downregulation of miR-218 was found to be associated with a history of cigarette smoking, but not human papilloma virus. Thus, we show for the first time that putative lung cancer-associated miRNAs can be identified from genome-wide arrayCGH datasets using a bioinformatics mapping approach, and report that miR-218 is a strong candidate tumour suppressing miRNA potentially involved in lung cancer

Reduced Expression of Brain-Enriched microRNAs in Glioblastomas Permits Targeted Regulation of a Cell Death Gene

Glioblastoma is a highly aggressive malignant tumor involving glial cells in the human brain. We used high-throughput sequencing to comprehensively profile the small RNAs expressed in glioblastoma and non-tumor brain tissues. MicroRNAs (miRNAs) made up the large majority of small RNAs, and we identified over 400 different cellular pre-miRNAs. No known viral miRNAs were detected in any of the samples analyzed. Cluster analysis revealed several miRNAs that were significantly down-regulated in glioblastomas, including miR-128, miR-124, miR-7, miR-139, miR-95, and miR-873. Post-transcriptional editing was observed for several miRNAs, including the miR-376 family, miR-411, miR-381, and miR-379. Using the deep sequencing information, we designed a lentiviral vector expressing a cell suicide gene, the herpes simplex virus thymidine kinase (HSV-TK) gene, under the regulation of a miRNA, miR-128, that was found to be enriched in non-tumor brain tissue yet down-regulated in glioblastomas, Glioblastoma cells transduced with this vector were selectively killed when cultured in the presence of ganciclovir. Using an in vitro model to recapitulate expression of brain-enriched miRNAs, we demonstrated that neuronally differentiated SH-SY5Y cells transduced with the miRNA-regulated HSV-TK vector are protected from killing by expression of endogenous miR-128. Together, these results provide an in-depth analysis of miRNA dysregulation in glioblastoma and demonstrate the potential utility of these data in the design of miRNA-regulated therapies for the treatment of brain cancers

Role of miR-10b in breast cancer metastasis

Ninety percent of cancer-related mortality is caused by metastasis. Current cancer treatments can control many primary tumors but rarely stop the metastatic spread. Accumulating evidence demonstrates that miRNAs are involved in cancer initiation and progression. Furthermore, several miRNAs have been found to regulate metastasis. In particular, recent studies provide the first functional evidence that overexpression of a specific miRNA, miR-10b, can contribute to the development of metastasis, which can be exploited therapeutically in treating breast cancer metastasis in mice. Further in-depth analysis should provide more precise evaluation of the roles, mechanisms, and therapeutic utility of this miRNA in breast cancer

Genomic analysis of microRNA time-course expression in liver of mice treated with genotoxic carcinogen N-ethyl-N-nitrosourea

<p>Abstract</p> <p>Background</p> <p>Dysregulated expression of microRNAs (miRNAs) has been previously observed in human cancer tissues and shown promise in defining tumor status. However, there is little information as to if or when expression changes of miRNAs occur in normal tissues after carcinogen exposure.</p> <p>Results</p> <p>To explore the possible time-course changes of miRNA expression induced by a carcinogen, we treated mice with one dose of 120 mg/kg <it>N</it>-ethyl-<it>N</it>-nitrosourea (ENU), a model genotoxic carcinogen, and vehicle control. The miRNA expression profiles were assessed in the mouse livers in a time-course design. miRNAs were isolated from the livers at days 1, 3, 7, 15, 30 and 120 after the treatment and their expression was determined using a miRNA PCR Array. Principal component analysis of the miRNA expression profiles showed that miRNA expression at post-treatment days (PTDs) 7 and 15 were different from those at the other time points and the control. The number of differentially expressed miRNAs (DEMs) changed over time (3, 5, 14, 32, 5 and 5 at PTDs 1, 3, 7, 15, 30 and 120, respectively). The magnitude of the expression change varied with time with the highest changes at PTDs 7 or 15 for most of the DEMs. In silico functional analysis of the DEMs at PTDs 7 and 15 indicated that the major functions of these ENU-induced DEMs were associated with DNA damage, DNA repair, apoptosis and other processes related to carcinogenesis.</p> <p>Conclusion</p> <p>Our results showed that many miRNAs changed their expression to respond the exposure of the genotoxic carcinogen ENU and the number and magnitude of the changes were highest at PTDs 7 to 15. Thus, one to two weeks after the exposure is the best time for miRNA expression sampling.</p