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

Genome-Wide Profiling Identified a Set of miRNAs that Are Differentially Expressed in Glioblastoma Stem Cells and Normal Neural Stem Cells

A major challenge in cancer research field is to define molecular features that distinguish cancer stem cells from normal stem cells. In this study, we compared microRNA (miRNA) expression profiles in human glioblastoma stem cells and normal neural stem cells using combined microarray and deep sequencing analyses. These studies allowed us to identify a set of 10 miRNAs that are considerably up-regulated or down-regulated in glioblastoma stem cells. Among them, 5 miRNAs were further confirmed to have altered expression in three independent lines of glioblastoma stem cells by real-time RT-PCR analysis. Moreover, two of the miRNAs with increased expression in glioblastoma stem cells also exhibited elevated expression in glioblastoma patient tissues examined, while two miRNAs with decreased expression in glioblastoma stem cells displayed reduced expression in tumor tissues. Furthermore, we identified two oncogenes, NRAS and PIM3, as downstream targets of miR-124, one of the down-regulated miRNAs; and a tumor suppressor, CSMD1, as a downstream target of miR-10a and miR-10b, two of the up-regulated miRNAs. In summary, this study led to the identification of a set of miRNAs that are differentially expressed in glioblastoma stem cells and normal neural stem cells. Characterizing the role of these miRNAs in glioblastoma stem cells may lead to the development of miRNA-based therapies that specifically target tumor stem cells, but spare normal stem cells

Identification and characterization of microRNAs expressed in the African malaria vector Anopheles funestus life stages using high throughput sequencing

Background: Over the past several years, thousands of microRNAs (miRNAs) have been identified in the genomes of various insects through cloning and sequencing or even by computational prediction. However, the number of miRNAs identified in anopheline species is low and little is known about their role. The mosquito Anopheles funestus is one of the dominant malaria vectors in Africa, which infects and kills millions of people every year. Therefore, small RNA molecules isolated from the four life stages (eggs, larvae, pupae and unfed adult females) of An. funestus were sequenced using next generation sequencing technology. Results: High throughput sequencing of four replicates in combination with computational analysis identified 107 mature miRNA sequences expressed in the An. funestus mosquito. These include 20 novel miRNAs without sequence identity in any organism and eight miRNAs not previously reported in the Anopheles genus but are known in non-anopheles mosquitoes. Finally, the changes in the expression of miRNAs during the mosquito development were determined and the analysis showed that many miRNAs have stage-specific expression, and are co-transcribed and co-regulated during development. Conclusions: This study presents the first direct experimental evidence of miRNAs in An. funestus and the first profiling study of miRNA associated with the maturation in this mosquito. Overall, the results indicate that miRNAs play important roles during the growth and development. Silencing such molecules in a specific life stage could decrease the vector population and therefore interrupt malaria transmission.IS

Landscape and variation of RNA secondary structure across the human transcriptome

In parallel to the genetic code for protein synthesis, a second layer of information is embedded in all RNA transcripts in the form of RNA structure. RNA structure influences practically every step in the gene expression program1. Yet the nature of most RNA structures or effects of sequence variation on structure are not known. Here we report the initial landscape and variation of RNA secondary structures (RSS) in a human family Trio, providing a comprehensive RSS map of human coding and noncoding RNAs. We identify unique RSS signatures that demarcate open reading frames, splicing junctions, and define authentic microRNA binding sites. Comparison of native deproteinized RNA isolated from cells versus refolded purified RNA suggests that the majority of the RSS information is encoded within RNA sequence. Over 1900 transcribed single nucleotide variants (~15 % of all transcribed SNVs) alter local RNA structure. We discover simple sequence and spacing rules that determine the ability of point mutations to impact RSS. Selective depletion of RiboSNitches versus structurally synonymous variants at precise locations suggests selection for specific RNA shapes at thousands of sites, including 3’UTRs, binding sites of miRNAs and RNA binding proteins genome-wide. These results highlight the potentially broa

MicroRNA Profile in CD8+ T-Lymphocytes from HIV-Infected Individuals: Relationship with Antiviral Immune Response and Disease Progression

Background The relationship between host microRNAs (miRNA), viral control and immune response has not yet been elucidated in the field of HIV. The aim of this study was to assess the differential miRNA profile in CD8+ T-cells between HIV-infected individuals who differ in terms of viral replication control and immune response. Methods miRNA profile from resting and CD3/CD28-stimulated CD8+ T-cells from uninfected individuals (HIV-, n = 11), Elite Controllers (EC, n = 15), Viremic Controllers (VC, n = 15), Viremic Progressors (VP, n = 13) and HIV-infected patients on therapy (ART, n = 14) was assessed using Affymetrix miRNA 3.1 arrays. After background correction, quantile normalization and median polish summarization, normalized data were fit to a linear model. The analysis comprised: resting samples between groups; stimulated samples between groups; and stimulated versus resting samples within each group. Enrichment analyses of the putative target genes were perfomed using bioinformatic algorithms. Results A downregulated miRNA pattern was observed when resting samples from all infected groups were compared to HIV-. A miRNA downregulation was also observed when stimulated samples from EC, ART and HIV- groups were compared to VP, being hsa-miR-4492 the most downregulated. Although a preferential miRNA downregulation was observed when stimulated samples were compared to the respective resting samples, VP presented a differential miRNA expression pattern. In fact, hsa-miR-155 and hsa-miR-181a were downregulated in VP whereas in the other groups, either an upregulation or no differences were observed after stimulation, respectively. Overall, functional enrichment analysis revealed that the predicted target genes were involved in signal transduction pathways, metabolic regulation, apoptosis, and immune response. Conclusions Resting CD8+ T-cells do not exhibit a differential miRNA expression between HIV-infected individuals but they do differ from non-infected individuals. Moreover, a specific miRNA pattern is present in stimulated CD8+ T-cells from VP which could reflect a detrimental pattern in terms of CD8+ T-cell immune response

A human herpesvirus miRNA attenuates interferon signaling and contributes to maintenance of viral latency by targeting IKKɛ

Type I interferon (IFN) signaling is the principal response mediating antiviral innate immunity. IFN transcription is dependent upon the activation of transcription factors IRF3/IRF7 and NF-κB. Many viral proteins have been shown as being capable of interfering with IFN signaling to facilitate evasion from the host innate immune response. Here, we report that a viral miRNA, miR-K12-11, encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) is critical for the modulation of IFN signaling and acts through targeting I-kappa-B kinase epsilon (IKKɛ). Ectopic expression of miR-K12-11 resulted in decreased IKKɛ expression, while inhibition of miR-K12-11 was found to restore IKKɛ expression in KSHV-infected cells. Importantly, expression of miR-K12-11 attenuated IFN signaling by decreasing IKKɛ-mediated IRF3/IRF7 phosphorylation and by inhibiting the activation of IKKɛ-dependent IFN stimulating genes (ISGs), allowing miR-K12-11 suppression of antiviral immunity. Our data suggest that IKKɛ targeting by miR-K12-11 is an important strategy utilized by KSHV to modulate IFN signaling during the KSHV lifecycle, especially in latency. We also demonstrated that IKKɛ was able to enhance KSHV reactivation synergistically with the treatment of 12-O-tetradecanoylphorbol 13-acetate. Moreover, inhibition of miR-K12-11 enhanced KSHV reactivation induced by vesicular stomatitis virus infection. Taken together, our findings also suggest that miR-K12-11 can contribute to maintenance of KSHV latency by targeting IKKɛ